EP3668497A1 - Procédés et compositions de traitement de maladies associées au cancer, à une inflammation ou à une réponse immunitaire - Google Patents

Procédés et compositions de traitement de maladies associées au cancer, à une inflammation ou à une réponse immunitaire

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Publication number
EP3668497A1
EP3668497A1 EP18845621.4A EP18845621A EP3668497A1 EP 3668497 A1 EP3668497 A1 EP 3668497A1 EP 18845621 A EP18845621 A EP 18845621A EP 3668497 A1 EP3668497 A1 EP 3668497A1
Authority
EP
European Patent Office
Prior art keywords
itaconate
unsubstituted
alkyl
substituted
cancer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP18845621.4A
Other languages
German (de)
English (en)
Other versions
EP3668497A4 (fr
Inventor
Maxim ARTYOMOV
Monika BAMBOUSKOVA
Vicky Lampropoulou
Roland Dolle
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Washington
Washington University in St Louis WUSTL
Original Assignee
University of Washington
Washington University in St Louis WUSTL
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Filing date
Publication date
Application filed by University of Washington, Washington University in St Louis WUSTL filed Critical University of Washington
Publication of EP3668497A1 publication Critical patent/EP3668497A1/fr
Publication of EP3668497A4 publication Critical patent/EP3668497A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present disclosure generally relates to methods and
  • compositions for the treatment of diseases associated with cancer, inflammation, or immune response can comprise administering an itaconate, malonate, or derivative thereof to a subject in need.
  • the disclosure provides administration of dimethyl itaconate to down-regulate Ikb- ⁇ induction, therefore useful in treatment for Ikb- ⁇ associated diseases, such as psoriasis, multiple sclerosis, and lymphoma (e.g., ABC subtype of DLBL lymphoma) or to reduce the extent of tissue injury in cardiovascular infarction.
  • Ikb- ⁇ associated diseases such as psoriasis, multiple sclerosis, and lymphoma (e.g., ABC subtype of DLBL lymphoma) or to reduce the extent of tissue injury in cardiovascular infarction.
  • One aspect of the present disclosure is directed to the provision of a composition comprising an itaconate, malonate, or derivative thereof and uses thereof.
  • the present disclosure provides for methods of treating Ikb- ⁇ associated disease, disorder, or conditions comprising administering a therapeutically effective amount of an itaconate, malonate, or derivative thereof to a subject.
  • the therapeutically effective amount reduces or prevents tumor growth, inflammation, or an immune response.
  • Another aspect of the present disclosure includes a method to suppress Ikb- ⁇ induction comprising administering an Ikb- ⁇ modulation agent.
  • Another aspect of the present disclosure includes a method of inhibiting tumor growth comprising administering an Ikb- ⁇ modulation agent.
  • the Ikb- ⁇ modulation agent comprises itaconate, itaconic acid, dimethyl itaconate (DI), 4-methyl itaconate, 3-(ethoxycarbonyl)but-3-enoic acid, 4-ethoxy-2- methylene-4-oxobutanoic acid, 4-octyl itaconate, dimethyl fumarate (DMF), diethyl malonate (DEM), dimethyl malonate, malonate, malonic acid, 2-methylenesuccinic acid, monoethylitaconate, 2-methyl fumaric acid, fuamaric acid, or a derivative or a
  • the itaconate, malonate, or a derivative thereof comprises a compound of formula I:
  • R 1 is hydrogen, unsubstituted or substituted alkyl; unsubstituted or substituted alkenes; or unsubstituted or substituted alkynes;
  • R 2 is hydrogen, unsubstituted or substituted alkyl; unsubstituted or substituted alkenes; or unsubstituted or substituted alkynes;
  • R 3 is hydrogen, unsubstituted or substituted alkyl; unsubstituted or substituted alkenes; or unsubstituted or substituted alkynes;
  • R 4 is hydrogen, unsubstituted or substituted alkyl; unsubstituted or substituted alkenes; or unsubstituted or substituted alkynes;
  • R 5 is hydrogen, unsubstituted or substituted alkyl; unsubstituted or substituted alkenes; or unsubstituted or substituted alkynes;
  • R 6 is hydrogen, unsubstituted or substituted alkyl; unsubstituted or substituted alkenes; or unsubstituted or substituted alkynes; and
  • R 7 is hydrogen, unsubstituted or substituted alkyl; unsubstituted or substituted alkenes; or unsubstituted or substituted alkynes; wherein
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 or R 7 can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; C 1-10 alkyl hydroxyl;
  • amine C 1-10 carboxylic acid; C 1-10 carboxyl; straight chain or branched C 1-10 alkyl, optionally containing unsaturation; a C 2-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched C 1-10 alkyl amine; heterocyclyl; heterocyclic amine; and aryl comprising a phenyl; heteroaryl containing from 1 to 4 N, O, or S atoms; unsubstituted phenyl ring; substituted phenyl ring; unsubstituted heterocyclyl; and substituted heterocyclyl, wherein
  • the unsubstituted phenyl ring or substituted phenyl ring can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; C 1-10 alkyl hydroxyl; amine; C 1-10 carboxylic acid; C 1-10 carboxyl; straight chain or branched C 1-10 alkyl, optionally containing unsaturation; straight chain or branched C 1-10 alkyl amine, optionally containing unsaturation; a C 2-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched C 1-10 alkyl amine; heterocyclyl; heterocyclic amine; aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, O, or S atoms; and the unsubstituted heterocyclyl or substituted heterocyclyl can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; C 1-10 alkyl hydroxyl;
  • FIG.1A, FIG.1B, FIG.1C, FIG.1D, FIG.1E, FIG.1F and FIG.1G show a series of images and graphs.
  • FIG.1A Expression of selected genes from RNA-Seq dataset generated from BMDMs treated or not with 250 ⁇ M DI for12 and stimulated with LPS 100 ng/ml for indicated times.
  • FIG.1B Structure of DI.
  • FIG.1C Nrf2 protein detection in whole cell lysates prepared from BMDM treated as in A and stimulated with LPS 100 ng/ml for indicated times.
  • FIG.1D BMDMs were treated with 250 ⁇ M DI for indicated times and production of intracellular ROS was determined by H2DCFDA detection by flow cytometry. MFI for H2DCFDA signal in samples is shown.
  • FIG.1E Total content of GSH in extracts prepared from BMDMs treated with 250 ⁇ M DI for indicated times.
  • FIG.1F GSH/GSSG ratio in extracts from BMDMs treated with 250 ⁇ M DI for indicated times.
  • FIG.1G BMDM were treated as in A and stimulated with 100 ng/ml LPS for 24 h. In some samples NAC was added at the time of DI addition (NAC_12) and in some samples NAC was added at the time of stimulation with LPS (NAC_0). Production of IL6 in cell supernatants was detected by ELISA.
  • FIG.2A, FIG.2B, FIG.2C, FIG.2 D, FIG.2E, FIG.2F, FIG.2G. FIG.2H, FIG.2I, FIG.2J and FIG.2K show a series of images and graphs comparing BMDM cells under treatment conditions.
  • FIG.2A BMDM were treated with DI 250 ⁇ M for 12h and stimulated with LPS for 30min. Cells were fixed, permeabilized and stained for nuclei (DAPI) p65 and F-actin (in green).
  • FIG.2B BMDM were treated with various DI concentration (100, 150, 250 ⁇ M) for 12h and stimulated with LPS for indicated times.
  • FIG.2C Phosphorylated IKK and total Ikb ⁇
  • FIG.2D BMDM were treated as in FIG.2A and stimulated with 100ng/ml LPS for indicated times.
  • Ikb ⁇ protein levels were detected by western blot.
  • GAPDH was used as loading control
  • FIG.2E BMDM were treated as in FIG.2A and stimulated with 100ng/ml LPS for indicated times.
  • Ikb ⁇ expression mRNA was analyzed by qPCR.
  • FIG.2F BMDM were treated as in FIG.2A and stimulated with 100ng/ml LPS for indicated times.
  • NAC was added at the time of DI addition (NAC_12) and in some samples NAC was added at the time of stimulation with LPS (NAC_0).
  • Ikb ⁇ protein levels were detected by western blot.
  • FIG.2G BMDM were treated as in FIG.2A and stimulated with LPS for indicated times.
  • 10 ⁇ M MG132 was added at the time of stimulation.
  • Ikb ⁇ protein levels were detected by western blot.
  • FIG.2H Human CD14+ monocytes were treated with 250 ⁇ M DI for 12 h and stimulated with 100ng/ml LPS for indicated times. Ikb ⁇ protein levels were detected by western blot.
  • FIG.2I Human CD14+ monocytes treated as in FIG.2G were stimulated with100ng/ml LPS for 24 h. Cytokine levels in cell supernatants were detected by ELISA.
  • FIG.2J GFP or GFP fused to 3-UTR sequence of Nfkbiz mRNA were introduced to BV2 cells by lentiviral transduction. Positive clones were selected by puromycin. Cells were then treated with 250 ⁇ M DI for 12 h and stimulated with 100 ng/ml LPS for 1h. GFP fluorescence was determined by flow cytometry.
  • FIG.2K BMDM were treated as in FIG.2A and stimulated with 100 ng/ml LPS for indicated times. Regnase-1 protein levels were detected by western blot.
  • FIG.3A and FIG.3B show a series of Western blots.
  • FIG.3A BMDM derived from WT or Nrf2 KO mice were treated with 250 ⁇ M DI for 12 h and stimulated with 100 ng/ml LPS for indicated times. Ikb- ⁇ protein levels were detected by western blot. GAPDH was used as loading control.
  • FIG.3B BMDMs were treated as in FIG.3A and STAT3 phosphorylation at Y705 or S727 sites was determined by western blot. GAPDH was used as loading control.
  • FIG.4B, FIG.4C, FIG.4D and FIG.4E show a series of graphs.
  • FIG.4A HaCat cells were treated with DI as indicated for 12h and then stimulated with 100ng/ml IL17A for indicated times. Ikb- ⁇ , STAT3 and STAT3
  • FIG.4B HaCat cells were treated as in A and cell viability was determined by propidium iodide staining and flow cytometry.
  • FIG.4C Human primary keratinocytes were treated with DI as indicated for 12h and then stimulated with 100ng/ml IL17 for indicated times. Ikb- ⁇ protein levels were detected by western blot. GAPDH was used as loading control.
  • FIG.4D Human primary keratinocytes were treated as in FIG.4C and cell viability was determined by propidium iodide staining and flow cytometry.
  • FIG. 4E Human primary keratinocytes were treated and stimulated as in FIG.4C and mRNA expression of selected genes was analyzed by qPCR.
  • FIG.5A and FIG.5B is a series of images and a graph.
  • DI was administered to mice daily, which led to significant improvement of the psoriatic pathology.
  • FIG.5B The same daily regimen of DI or vehicle (ve) administration was used in vivo as in the EAE model of the mice and found that DI prevented clinical development of EAE.
  • FIG.6 is a bar graph showing DLBCL cell lines cultured at the presence of indicated concentrations of DI. Media containing DI was refreshed every day for four days in total. Cell viability was determined by resazurin assay at the day four.
  • FIG.7A and FIG.7B is a series of Western blots and bar graphs.
  • FIG.7A BMDM were treated with DMF at different concentrations for 12 h and stimulated with 100 ng/ml LPS for indicated times. Ikb- ⁇ and pro-IL1 ⁇ protein levels and STAT3 activation were determined by western blot. GAPDH was used as loading control.
  • FIG.7B BMDM were treated as in FIG.7A and stimulated with 100 ng/ml LPS for indicated times. Cytokine production in cell supernatants was determined by ELISA.
  • FIG.8A, FIG.8B, FIG.8C, FIG.8D, FIG.8E, FIG.8F, FIG.8G and FIG.8H show a series of graphs and images including the structure of DI and comparison transcriptional profiles.
  • FIG.8A Structure of DI.
  • FIG.8B Comparison of transcriptional profiles of cKeap KO BMDMs and BMDMs treated with 250 ⁇ M DI for 12 h.
  • FIG.8C Triggering of Nrf2 expression and expression of Nrf2-targets (Nqo1, HO-1) in BMDMs treated as in B.
  • FIG.8D Analysis of intracellular DI uptake and identification of DI-glutathione adduct.
  • FIG.8E ROS production measured by detection of CM- H2DCFDA in BV2 cells treated with DI as in B.
  • FIG.8F Glutathione depletion and GSH/GSSG ratio (FIG.8G) in BMDMs treated with DI as in FIG.8B.
  • FIG.8H N- acetylcysteine (NAC) neutralizes effect of DI on cytokine production.
  • BMDMs were treated with 250 ⁇ M DI and stimulated with LPS for 12 h. In some samples NAC was added simultaneously with DI.
  • FIG.9A, FIG.9B and FIG.9C show a series of Western blot images and fluorescent microscope images.
  • FIG.9A BMDMs were treated with 250 ⁇ M DI for 12 h and stimulated with LPS. Loss of IRAK1 detection upon LPS refers to its K63 ubiquitination that correlates with activation. DI affected phosphorylation of IKK in time-dependent manner.
  • FIG.9B Degradation of Ikb ⁇ is also not affected by DI treatment.
  • FIG.9C BMDMs were treated with DI as in A and fixed, permeabilized and stained for p65, F-Actin and nuclei. DI pretreatment does not inhibit nuclear
  • FIG.10A, FIG.10B, FIG.10C, FIG.10D, FIG.10E, FIG.10F, FIG. 10G and FIG.10H show a series of Western blots images and graphs.
  • FIG.10A Detection of Ikb ⁇ expression in BMDMs treated with DI for 12 h and stimulated with LPS.
  • FIG.10B Dose dependent effect of DI on cytokine production in BMDMs pretreated with DI for 12 h and stimulated with LPS for 4 h.
  • FIG.10C Dose dependent inhibition of Ikb ⁇ expression in in cell treated as in FIG.10B and stimulated with LPS for 1 h.
  • FIG.10D Nfkbiz mRNA expression in Ikb ⁇ protein expression in cells treated as in FIG.10C.
  • FIG.10E and FIG.10G Diminished Ikb ⁇ protein expression is not due to the proteasomal degradation as shown by treatment of cells with MG132 or due to lysosomal/autophagy degradation as shown by treatment of cells with BafalomycinA (BafA).
  • FIG.10F Dimethyl malonate (DM), a succinyl dehydrogenase inhibitor, does not affect Ikb ⁇ expression.
  • BMDMs were treated with 10 mM DM for 12 h and stimulated with LPS.
  • FIG.10H Detection of Ikb ⁇ expression in human CD14+ monocytes treated with DI for 12h and stimulated with LPS.
  • FIG.11A, FIG.11B, FIG.11C and FIG.11D show a series of images and graphs.
  • FIG.11A Ikb ⁇ expression in BMDMs treated with 250 ⁇ M DI for 12h and stimulated with LPS. In some samples N-acetylcysteine was added
  • FIG.11B Ikb ⁇ expression in Nrf2 deficient BMDMs. Cell were treated with 250 ⁇ M DI for 12 h prior to LPS stimulation.
  • FIG.11C Transcriptional comparison of DI treated WT and Nrf2 KO BMDM. Left panel shows signature pathways up-regulated and down-regulated by DI independent of Nrf2.
  • FIG.11D Most significantly up-regulated genes by DI in Nrf2- independent manner.
  • FIG.12A, FIG.12B, FIG.12C, FIG.12D, FIG.12E and FIG.12F show a series of Western blots and bar graphs.
  • FIG.12A shows Ikb ⁇ expression in primary mouse keratinocytes that were treated with DI for 12h and then stimulated with IL-17A (100 ng/ml).
  • FIG.12B Viability of mouse keratinocytes treated as in FIG.12A.
  • FIG.12C qPCR analysis of gene expression in mouse keratinocytes treated as in FIG. 12A.
  • FIG.12D Ikb ⁇ expression in primary human keratinocytes that were treated with DI for 12h then stimulated with IL-17A (100 ng/ml).
  • FIG.12E Viability of mouse keratinocytes treated as in FIG.12D.
  • FIG.12F qPCR analysis of gene expression in mouse keratinocytes treated as in FIG.12D.
  • FIG.13A and FIG.13B is a series of images and bar graphs.
  • FIG. 13A Bl6 mice were injected i.p. with a dose of DI and imiquimod (IMQ) was applied topically on ear skin daily for 7 days. Sections of imiquimod-treated ears from mice following 5 d of treatment are shown.
  • FIG.13B qPCR analysis of gene expression in skin tissue of mice treated as in FIG.13A.
  • FIG.14A, FIG.14B, FIG.14C, FIG.14D, FIG.14E, FIG.14F, FIG. 14G, FIG.14H and FIG.14I depict a series of images and graphs showing that DI induces electrophilic stress in macrophages.
  • FIG.14A Nrf2 response genes in DI treated BMDMs.
  • FIG.14B and FIG.14C Western blot of Nrf2 and Nrf2 targets in DI treated or LPS-stimulated BMDMs.
  • FIG.14D Structures of DI- or Ita-GSH.
  • Western blots are representatives of 3 experiments. Two-tailed t-test.
  • FIG.15A, FIG.15B, FIG.15C, FIG.15D, FIG.15E, FIG.15F, FIG. 15G, FIG.15H, FIG.15I and FIG.15J depict a series of images and graphs showing DI- GSH and Ita-GSH detection and electrophilic stress response. (FIG.15A)
  • FIG.15B DI reacting with thiol group in Michael reaction.
  • FIG.15E DI-GSH conjugate levels in
  • FIG.15J Western blot of HO-1 in BMDMs treated with DMF. Representative of 3 experiments. Two-tailed t test.
  • FIG.16A, FIG.16B, FIG.16C, FIG.16D, FIG.16E, FIG.16F, FIG. 16G, FIG.16H and FIG.16I show a series of images and graphs showing DI inhibits LPS-mediated I ⁇ B ⁇ induction.
  • FIG.16A mRNA expression in LPS-stimulated BMDMs. Representative of 2 experiments.
  • FIG.16B, FIG.16C, FIG.16G, FIG.16H, FIG.16I, and FIG.16J Western blot of I ⁇ B ⁇ expression in BMDMs treated with DI, DMF, MI (FIG.16H, 5 ⁇ M, FIG.16J, 10 ⁇ M) or 3MI, LPS for 1 h or as indicated.
  • FIG.16E Scheme of DI action.
  • FIG.16F Structures of DMF and itaconate derivatives.
  • FIG.16I Western blot of I ⁇ B ⁇ expression in BMDMs tolerized in presence of BSO. Western blots are representative of 3
  • FIG.17A, FIG.17B, FIG.17C, FIG.17D, FIG.17E, FIG.17F, FIG. 17G, FIG.17H, FIG.17I, FIG.17J, FIG.17K, FIG.17L and FIG.17M show a series of images and graphs showing that DI downregulates secondary transcriptional response to TLR stimulation.
  • FIG.17A Western blot of I ⁇ B ⁇ expression in WT or Nfkbiz-/- BMDMs stimulated with LPS.
  • FIG.17C RNA- seq analysis of BMDMs treated with DI and stimulated with LPS and IFN- ⁇ .
  • FIG.17E Western blot of I ⁇ B ⁇ expression in DI-treated BMDMs stimulated with LPS 1 h.
  • FIG.17F mRNA expression in human blood monocytes treated with DI and stimulated with LPS.
  • FIG.17G Western blot of I ⁇ B ⁇ expression in human blood monocytes treated DI and stimulated with LPS.
  • FIG.17H and FIG.17I Western blot of FIG.17H, I ⁇ B ⁇ or FIG.17I, IRAK1 expression and IKK phosphorylation in BMDMs treated with DI and stimulated with LPS.
  • FIG.17J p65 localization in DI-treated, LPS-stimulated BMDMs.
  • DAPI nuclei. Bars 25 ⁇ M.
  • FIG.17K Western blot of I ⁇ B ⁇ expression in BMDMs treated with DI in presence of EtGSH and stimulated with LPS for 1 h.
  • FIG.17L Western blot of I ⁇ B ⁇ expression in human blood monocytes treated with DI in presence of EtGSH and stimulated with LPS for 1 h.
  • FIG.17A Representative data in FIG.17A from 2 experiments; FIG.17E, FIG.17H, FIG.17I, and FIG.17K are from 3 experiments; FIG.17F and FIG.17G are representative data of 3 donors; FIG.17I, 2 donors. Two-tailed t test.
  • FIG.18A, FIG.18B, FIG.18C, FIG.18D, FIG.18E, FIG.18F, FIG. 18G and FIG.18H show a series of images and graphs showing DI regulates I ⁇ B ⁇ on post-transcriptional level.
  • FIG.18B Western blot of I ⁇ B ⁇ expression in BMDMs treated with DI and stimulated with LPS for 1 h. MG132 or bafilomycin A (BafA) were added 30 min before LPS stimulation.
  • FIG.18C Nfkbiz 3’UTR reporter expressing GFP in BV2 cells treated with DI (250 ⁇ M) for 12 h and stimulated with LPS for 1 h. EMPTY vector expressing GFP only. GFP expression determined by flow cytometry.
  • FIG.18D Western blot of phosphorylated and total eIF2 ⁇ in DI treated BMDMs.
  • FIG.18E Western blot of nascent protein synthesis detected using biotin-alkyne Click chemistry in BMDMs treated with DI and stimulated with LPS for 1h. Same membrane was reprobed for I ⁇ B ⁇ . Representative of 2
  • FIG.18F Densitometric quantification of biotin signal in membrane in FIG.18E.
  • FIG.18G Log fold change of proteomic signal in unstimulated versus LPS stimulated cells.
  • FIG.18H Log fold change of transcript versus protein.
  • FIG.18B, FIG.18C, and FIG.18D Representative data from 3 experiments.
  • FIG.19A, FIG.19B, FIG.19C, FIG.19D, FIG.19E and FIG.19F show a series of images and graphs showing BSO potentiates inhibitory effect of DI.
  • FIG.19A Western blot of Nrf2 expression in BMDMs were treated with BSO or DI.
  • FIG.19F Western blot of I ⁇ B ⁇ expression in BMDMs tolerized with LPS in presence of BSO for 18 h and restimulated for 1 h (see e.g., FIG.16L), asterisk shows different exposure. Western blot data are representative of 3 experiments. Two-tailed t test.
  • FIG.20A, FIG.20B, FIG.20C, FIG.20D, FIG.20E, FIG.20F, FIG. 20G and FIG.20H show a series of images and graphs showing that DI induces Nrf2- independent response and inhibits IL-6/I ⁇ B ⁇ axis via ATF3.
  • FIG.20A and FIG.20F Western blot of I ⁇ B ⁇ expression;
  • FIG.20C Genes regulated by DI independently of Nrf2.
  • FIG.20D Transcriptional comparison of Atf3-/- and WT BMDMs and enrichment of DI signature.
  • FIG.20E and FIG.20H Western blot of ATF3 expression in BMDMs (FIG.20E) DI- treated; (FIG.20H) tolerized in presence of BSO. Western blot data are representative of 3 experiments. Two-tailed t-test.
  • FIG.21A, FIG.21B, FIG.21C, FIG.21D, FIG.21E, FIG.21F, FIG. 21G and FIG.21H show a series of images and graphs showing the Nrf2-independent action of DI.
  • FIG.21A Western blot of I ⁇ B ⁇ expression in WT or Nrf2-/- BMDMs treated with DI and stimulated with LPS for 1h.
  • FIG.21B Western blot of p62 and HO- 1 in WT or Nrf2-/- BMDMs treated with DI and stimulated with LPS.
  • FIG.21C Western blot of I ⁇ B ⁇ expression in WT and p62-deficient BMDMs treated with DI and stimulated with LPS.
  • FIG.21D Western blot of I ⁇ B ⁇ expression in WT and Hmox1-deficient BMDMs treated with DI and stimulated with LPS.
  • FIG.21E Transcriptional comparison of Nrf2-/- and WT BMDMs treated with DI and GSEA statistics for unfolded protein response (UPR) and IFN- ⁇ pathways.
  • FIG.21F Pathways regulated by DI in Nrf2-/- independent manner. Gene ranks, normalized enrichment score (NES), P and adjusted P (padj) are shown.
  • FIG.21G and FIG.21H Western blot of FIG.21G, Nrf2 expression or FIG.21H, phosphorylated and total eIF2 ⁇ in DI-treated WT or Atf3-/- BMDMs.
  • FIG. 21I, FIG.21J, and FIG.21K Western blot of ATF3 in cells treated with DI in
  • FIG.21I and FIG.21J BMDMs
  • FIG.21K human blood monocytes.
  • Data in FIG.21A, FIG.21G, FIG.21I, and FIG.21J are representatives from 3 experiments, data in FIG.21B, FIG.21C, and FIG.21H from 2 experiments, data in FIG.21D were performed in one experiment and FIG.21K are representative data from 2 donors.
  • FIG.22A, FIG.22B, FIG.22C, FIG.22D, FIG.22E, FIG.22F, FIG. 22G and FIG.22H show a series of images and graphs showing that DI inhibits IL-17- mediated I ⁇ B ⁇ induction in keratinocytes and ameliorates psoriatic pathology.
  • FIG.22A and FIG.22B Western blot of I ⁇ B ⁇ expression in DI-treated, IL-17A-stimulated primary keratinocytes. Representative of 3 mice/donors.
  • FIG.22E DI administration in psoriasis model.
  • FIG.22F Ear histology. Bars 100 ⁇ M. Representative of 6 mice in 2 experiments.
  • FIG.23 shows set of graphs showing keratinocyte viability after DI treatment.
  • Mouse or human primary keratinocytes were treated with DI for 12 h and viability was determined by propidium iodide staining and flow cytometry. Percentage of PI negative cells is shown. Representative of 2 mice/donors.
  • FIG.24A, FIG.24B and FIG.24C show a series of images and graphs showing the lack of in vivo toxicity of DI.
  • FIG.24A Scheme of DI administration for analysis of SDH activity in heart and liver.
  • FIG.24C Western blot of SDH and GAPDH in mitochondrial and cytoplasmic fractions from heart and liver of mice treated as in FIG.24A.
  • FIG.25 shows a series of flow cytometry gating. Dead cells and debris were gated out based on FSC and SSC. Next, single cells were gated based on FSC. Final dot plots indicate FCS versus GFP signal. GFP negative cells (non- transduced BV2 as shown) were used to set the gate for GFP positive cells. DETAILED DESCRIPTION
  • compositions comprising itaconate, malonate or derivatives thereof and methods of use. Applicants have discovered that itaconate, malonate and derivatives thereof can modulate immune response, modulate
  • Ikbz/Il17 associated diseases such as: 1) auto-inflammatory diseases such as psoriasis and multiple sclerosis, as well as 2) Ikb ⁇ -dependent tumors such as ABC subtype of DLBCL.
  • compositions Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules of the compound are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary.
  • One aspect of the present disclosure encompasses itaconate, malonate, and derivatives thereof. Itaconate, malonate, or derivatives thereof may be modified to improve potency, bioavailability, solubility, stability, handling properties, or a combination thereof, as compared to an unmodified version.
  • a composition of the invention comprises modified itaconate, malonate, or derivatives thereof.
  • a composition of the invention comprises a prodrug of a itaconate, malonate, or derivatives thereof.
  • a composition of the invention may optionally comprise one or more additional drug or therapeutically active agent in addition to the itaconate, malonate, or derivatives thereof.
  • a composition of the invention may further comprise a pharmaceutically acceptable excipient, carrier, or diluent.
  • a composition of the invention may contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents, or antioxidants.
  • the compounds detailed herein include compounds comprising an itaconate, structure as diagrammed below. Itaconate is a non-peptide synthetic. Its chemical elements are expressed as C 5 H 4 O 4 -2 , and its synthesis is known. For example, dry distillation of citric acid affords itaconic anhydride, which undergoes hydrolysis to itaconic acid. Itaconic acid is manufactured commercially.
  • the compounds detailed herein include compounds comprising malonate, or Propanedioate, structure as diagrammed below.
  • Malonate is a non-peptide synthetic molecule with a molecular weight of 102.045 g/mol. Its chemical elements are expressed as C 3 H 2 O -2
  • an“itaconate derivative” or“malonate derivate” may be any derivative known in the art, a derivative of Formula (I) or Formula (II). Itaconate derivatives and malonate derivates are known in the art.
  • an itaconate or malonate derivative can be a dimethyl itaconate (DI), dimethyl fumarate (DMF), diethyl malonate (DEM), or a derivative thereof.
  • R 1 , R 2 , R 3 , and R 4 are each independently selected from the group consisting of hydrogen, deuterium , unsubstituted or substituted alkyl;
  • R 1 , R 2 , R 3 , and R 4 can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; C 1-10 alkyl hydroxyl; amine; C 1-10 carboxylic acid; C 1-10 carboxyl; straight chain or branched C 1-10 alkyl, optionally containing unsaturation; a C 2-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched C 1-10 alkyl amine; heterocyclyl; heterocyclic amine; and aryl comprising a phenyl; heteroaryl containing from 1 to 4 N, O, or S atoms; unsubstituted phenyl ring; substituted phenyl ring; unsubstituted heterocyclyl; and substituted heterocyclyl, wherein
  • the unsubstituted phenyl ring or substituted phenyl ring can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; C 1-10 alkyl hydroxyl; amine; C 1-10 carboxylic acid; C1- 10carboxyl; straight chain or branched C 1-10 alkyl, optionally containing unsaturation; straight chain or branched C1-10alkyl amine, optionally containing unsaturation; a C 2-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched C 1-10 alkyl amine; heterocyclyl;
  • heterocyclic amine aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, O, or S atoms;
  • the unsubstituted heterocyclyl or substituted heterocyclyl can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; C 1-10 alkyl hydroxyl; amine; C 1-10 carboxylic acid; C 1-10 carboxyl; straight chain or branched C 1-10 alkyl, optionally containing unsaturation; straight chain or branched C 1-10 alkyl amine, optionally containing unsaturation; a C 2-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; heterocyclyl; straight chain or branched C 1-10 alkyl amine;
  • heterocyclic amine and aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, O, or S atoms.
  • the dashed lines can indicate a bond, a double bond, or a delocalized bond.
  • a compound of Formula (I) comprises any of the preceding compounds of Formula (I), wherein R 1 may be selected from the group consisting of hydrogen, deuterium, C 1-10 alkyl or CH 3 .
  • a compound of Formula (I) comprises any of the proceeding compounds of Formula (I), wherein R 1 is H.
  • a compound of Formula (I) comprises any of the proceeding compounds of Formula (I), wherein R 1 is a C 1-10 alkyl.
  • a compound of Formula (I) comprises any of the proceeding compounds of Formula (I), wherein R 1 is a CH 3 .
  • a compound of Formula (I) comprises any of the proceeding compounds of Formula (I), wherein R 1 is a C 2 H 5 .
  • a compound of Formula (I) comprises any of the preceding compounds of Formula (I), wherein R 2 may be selected from the group consisting of hydrogen, deuterium CH 2 , C 1-10 alkyl, or CH 3 .
  • a compound of Formula (I) comprises any of the proceeding compounds of Formula (I), wherein R 2 is hydrogen.
  • a compound of Formula (I) comprises any of the proceeding compounds of Formula (I), wherein R 2 is a C 1-10 alkyl.
  • a compound of Formula (I) comprises any of the proceeding compounds of Formula (I), wherein R 2 is a CH 3 .
  • a compound of Formula (I) comprises any of the proceeding compounds of Formula (I), wherein R 2 is a CH 2 .
  • a compound of Formula (I) comprises any of the preceding compounds of Formula (I), wherein R 3 may be selected from the group consisting of hydrogen, deuterium C 1-10 alkyl, CH 3 , or C 8 H 17 .
  • a compound of Formula (I) comprises any of the proceeding compounds of Formula (I), wherein R 3 is selected is hydrogen.
  • a compound of Formula (I) comprises any of the proceeding compounds of Formula (I), wherein R 3 is a C 1-10 alkyl.
  • a compound of Formula (I) comprises any of the proceeding compounds of Formula (I), wherein R 3 is a CH 3 .
  • a compound of Formula (I) comprises any of the proceeding compounds of Formula (I), wherein R 3 is C 8 H 17 .
  • a compound of Formula (I) comprises any of the preceding compounds of Formula (I), wherein R 4 may be selected from the group consisting of hydrogen, deuterium, C 1-10 alkyl, CH 2 or CH 3 .
  • a compound of Formula (I) comprises any of the proceeding compounds of Formula (I), wherein R 4 is hydrogen.
  • a compound of Formula (I) comprises any of the proceeding compounds of Formula (I), wherein R 4 is a C 1-10 alkyl.
  • a compound of Formula (I) comprises any of the proceeding compounds of Formula (I), wherein R 4 is CH 3 .
  • a compound of Formula (I) comprises any of the proceeding compounds of Formula (I), wherein R 4 is a CH 2 .
  • R 5 , R 6 , and R 7 are each independently selected from the group consisting of hydrogen, deuterium , unsubstituted or substituted alkyl; unsubstituted or substituted alkenes; or unsubstituted or substituted alkynes;
  • R 5 , R 6 , and R 7 can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; C 1-10 alkyl hydroxyl; amine; C 1-10 carboxylic acid; C 1-10 carboxyl; straight chain or branched C 1-10 alkyl, optionally containing unsaturation; a C 2-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched C 1-10 alkyl amine; heterocyclyl; heterocyclic amine; and aryl comprising a phenyl; heteroaryl containing from 1 to 4 N, O, or S atoms; unsubstituted phenyl ring; substituted phenyl ring; unsubstituted heterocyclyl; and substituted heterocyclyl, wherein
  • the unsubstituted phenyl ring or substituted phenyl ring can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; C 1-10 alkyl hydroxyl; amine; C 1-10 carboxylic acid; C1- 10carboxyl; straight chain or branched C 1-10 alkyl, optionally containing
  • heterocyclic amine aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, O, or S atoms;
  • the unsubstituted heterocyclyl or substituted heterocyclyl can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; C 1-10 alkyl hydroxyl; amine; C 1-10 carboxylic acid; C 1-10 carboxyl; straight chain or branched C 1-10 alkyl, optionally containing
  • heterocyclic amine and aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, O, or S atoms.
  • the dashed lines can indicate a bond, a double bond, or a delocalized bond.
  • a compound of Formula (II) comprises any of the preceding compounds of Formula (II), wherein R 5 may be selected from the group consisting of hydrogen, deuterium CH 2 , C 1-10 alkyl, or CH 3 .
  • a compound of Formula (II) comprises any of the proceeding compounds of Formula (II), wherein R 5 is hydrogen.
  • a compound of Formula (II) comprises any of the proceeding compounds of Formula (II), wherein R 5 is a C 1-10 alkyl.
  • a compound of Formula (II) comprises any of the proceeding compounds of Formula (II), wherein R 5 is a CH 3 .
  • a compound of Formula (II) comprises any of the preceding compounds of Formula (II), wherein R 6 may be selected from the group consisting of hydrogen, deuterium C 1-10 alkyl, or CH 3 .
  • a compound of Formula (II) comprises any of the proceeding compounds of Formula (II), wherein R 6 is selected is hydrogen.
  • a compound of Formula (II) comprises any of the proceeding compounds of Formula (II), wherein R 6 is a C 1-10 alkyl.
  • a compound of Formula (II) comprises any of the proceeding compounds of Formula (II), wherein R 6 is CH 3 .
  • a compound of Formula (II) comprises any of the preceding compounds of Formula (II), wherein R 7 may be selected from the group consisting of hydrogen, deuterium C 1-10 alkyl, or CH 3 .
  • a compound of Formula (II) comprises any of the proceeding compounds of Formula (II), wherein R 7 is selected is hydrogen.
  • a compound of Formula (II) comprises any of the proceeding compounds of Formula (II), wherein R 7 is a C 1-10 alkyl.
  • a compound of Formula (II) comprises any of the proceeding compounds of Formula (II), wherein R 7 is CH 3 .
  • a compound of the disclosure comprises Formula (I) or Formula (II) as shown below:
  • the itaconate, malonate, or derivative thereof is not dimethyl fumaric acid.
  • the present disclosure also provides pharmaceutical compositions.
  • the pharmaceutical composition comprises itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II), as an active ingredient, and at least one pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient may be a diluent, a binder, a filler, a buffering agent, a pH modifying agent, a disintegrant, a dispersant, a preservative, a lubricant, taste-masking agent, a flavoring agent, or a coloring agent.
  • the amount and types of excipients utilized to form pharmaceutical compositions may be selected according to known principles of pharmaceutical science.
  • a composition of the invention may optionally comprise one or more additional drug or therapeutically active agent in addition to the itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II).
  • additional drug or therapeutically active agent induces anti-inflammatory effects.
  • the secondary agent is an antibody.
  • the secondary agent is selected from a corticosteroid, a non-steroidal anti-inflammatory drug (NSAID), an intravenous immunoglobulin, a tyrosine kinase inhibitor, a fusion protein, a monoclonal antibody directed against one or more pro-inflammatory cytokines, a chemotherapeutic agent and a combination thereof.
  • NSAID non-steroidal anti-inflammatory drug
  • the secondary agent may be a glucocorticoid, a corticosteroid, a non-steroidal anti- inflammatory drug (NSAID), a phenolic antioxidant, an anti-proliferative drug, a tyrosine kinase inhibitor, an anti-IL-5 or an IL5 receptor monoclonal antibody, an anti-IL-13 or an anti-IL-13 receptor monoclonal antibody, an IL-4 or an IL-4 receptor monoclonal antibody, an anti IgE monoclonal antibody, a monoclonal antibody directed against one or more pro-inflammatory cytokines, a TNF- ⁇ inhibitor, a fusion protein, a
  • NSAID non-steroidal anti- inflammatory drug
  • the chemotherapeutic agent or a combination thereof.
  • the chemotherapeutic agent or a combination thereof.
  • anti-inflammatory drugs include, but are not limited to, alclofenac, alclometasone dipropionate, algestone acetonide, alpha amylase, amcinafal, amcinafide, amfenac sodium, amiprilose hydrochloride, anakinra, anirolac, anitrazafen, apazone, balsalazide disodium, bendazac, benoxaprofen, benzydamine hydrochloride, bromelains, broperamole, budesonide, carprofen, cicloprofen, cintazone, cliprofen, clobetasol propionate, clobetasone butyrate, clopirac, cloticasone propionate, cormethasone acetate, cortodoxone, curcumin, deflazacort, desonide, desoximetasone, dexamethasone dipropionate,
  • indomethacin indomethacin sodium, indoprofen, indoxole, intrazole, isoflupredone acetate, isoxepac, isoxicam, ketoprofen, lofemizole hydrochloride, lomoxicam, loteprednol etabonate, lysofylline, meclofenamate sodium, meclofenamic acid, meclorisone dibutyrate, mefenamic acid, mesalamine, meseclazone,
  • methylprednisolone suleptanate momiflumate, nabumetone, naproxen, naproxen sodium, naproxol, nimazone, olsalazine sodium, orgotein, orpanoxin, oxaprozin, oxyphenbutazone, paranyline hydrochloride, pentosan polysulfate sodium,
  • the tyrosine kinase inhibitor is imatinib.
  • the anti-IL-5 monoclonal antibody is mepolizumab or reslizumab.
  • the IL-5 receptor monoclonal antibody is
  • the anti-IL-4 monoclonal antibody is dulipumab.
  • the anti IgE monoclonal antibody is omalizumab.
  • the TNF- ⁇ inhibitor is infliximab, adalimumab, certolizumab pegol, or golimumab.
  • the secondary agent is a drug used to treat heart failure such as a beta-blocker, an ACE-inhibitor, an angiotensin receptor blocker (ARB), a neprilisine inhibitor or an aldosterone antagonist.
  • the excipient may be a diluent.
  • the diluent may be compressible (i.e., plastically deformable) or abrasively brittle.
  • suitable compressible diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose esters (i.e., acetate and butyrate mixed esters), ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, corn starch, phosphated corn starch, pregelatinized corn starch, rice starch, potato starch, tapioca starch, starch-lactose, starch-calcium carbonate, sodium starch glycolate, glucose, fructose, lactose, lactose monohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol, xylito
  • the excipient may be a binder.
  • Suitable binders include, but are not limited to, starches, pregelatinized starches, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C 12 -C 18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, polypeptides, oligopeptides, and combinations thereof.
  • the excipient may be a filler.
  • suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and
  • the filler may be calcium sulfate, both di- and tri-basic, starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, dibasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starches, lactose, sucrose, mannitol, or sorbitol.
  • the excipient may be a buffering agent.
  • suitable buffering agents include, but are not limited to, phosphates, carbonates, citrates, tris buffers, and buffered saline salts (e.g., Tris buffered saline or phosphate buffered saline).
  • the excipient may be a pH modifier.
  • the pH modifying agent may be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.
  • the excipient may be a disintegrant.
  • the disintegrant may be non-effervescent or effervescent.
  • Suitable examples of non- effervescent disintegrants include, but are not limited to, starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pecitin, and tragacanth.
  • suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.
  • the excipient may be a dispersant or dispersing enhancing agent.
  • Suitable dispersants may include, but are not limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose.
  • the excipient may be a
  • preservatives include antioxidants, such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate, citric acid, sodium citrate; chelators such as EDTA or EGTA; and antimicrobials, such as parabens, chlorobutanol, or phenol.
  • antioxidants such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate, citric acid, sodium citrate
  • chelators such as EDTA or EGTA
  • antimicrobials such as parabens, chlorobutanol, or phenol.
  • the excipient may be a lubricant.
  • suitable lubricants include minerals such as talc or silica; and fats such as vegetable stearin, magnesium stearate, or stearic acid.
  • the excipient may be a taste-masking agent.
  • Taste-masking materials include cellulose ethers; polyethylene glycols; polyvinyl alcohol; polyvinyl alcohol and polyethylene glycol copolymers; monoglycerides or triglycerides; acrylic polymers; mixtures of acrylic polymers with cellulose ethers;
  • the excipient may be a flavoring agent.
  • Flavoring agents may be chosen from synthetic flavor oils and flavoring aromatics and/or natural oils, extracts from plants, leaves, flowers, fruits, and combinations thereof.
  • the excipient may be a coloring agent.
  • Suitable color additives include, but are not limited to, food, drug and cosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drug and cosmetic colors (Ext. D&C).
  • the weight fraction of the excipient or combination of excipients in the composition may be about 99% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1% or less of the total weight of the composition.
  • compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety.
  • Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
  • formulation refers to preparing a drug in a form suitable for administration to a subject, such as a human.
  • a “formulation” can include pharmaceutically acceptable excipients, including diluents or carriers.
  • pharmaceutically acceptable can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of
  • pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States
  • pharmaceutically acceptable excipient can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents.
  • solvents dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents.
  • the use of such media and agents for pharmaceutical active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN:
  • Supplementary active ingredients can also be incorporated into the compositions.
  • a “stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 oC and about 60 oC, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.
  • the formulation should suit the mode of administration.
  • the agents of use with the current disclosure can be formulated by known methods for
  • a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, and rectal.
  • the individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents.
  • biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic or other physical forces.
  • Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency.
  • Controlled-release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects.
  • Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time.
  • the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body.
  • the controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
  • compositions can be formulated into various dosage forms and administered by a number of different means that will deliver a therapeutically effective amount of the active ingredient.
  • Such compositions can be administered orally (e.g. inhalation), parenterally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired.
  • Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, or intrasternal injection, or infusion techniques. Formulation of drugs is discussed in, for example, Gennaro, A. R., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (18th ed, 1995), and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, N.Y. (1980).
  • a composition may be a food supplement or a composition may be a cosmetic.
  • Solid dosage forms for oral administration include capsules, tablets, caplets, pills, powders, pellets, and granules.
  • the active ingredient is ordinarily combined with one or more pharmaceutically acceptable excipients, examples of which are detailed above.
  • Oral preparations may also be administered as aqueous suspensions, elixirs, or syrups. For these, the active
  • the ingredient may be combined with various sweetening or flavoring agents, coloring agents, and, if so desired, emulsifying and/or suspending agents, as well as diluents such as water, ethanol, glycerin, and combinations thereof.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • the preparation may be an aqueous or an oil-based solution.
  • Aqueous solutions may include a sterile diluent such as water, saline solution, a pharmaceutically acceptable polyol such as glycerol, propylene glycol, or other synthetic solvents; an antibacterial and/or antifungal agent such as benzyl alcohol, methyl paraben, chlorobutanol, phenol, thimerosal, and the like; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as etheylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and/or an agent for the adjustment of tonicity such as sodium chloride, dextrose, or a
  • a sterile diluent such as water, saline solution, a pharmaceutically acceptable polyol such as glycerol, propylene glycol, or other synthetic solvents
  • compositions such as mannitol or sorbitol.
  • the pH of the aqueous solution may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide.
  • Oil-based solutions or suspensions may further comprise sesame, peanut, olive oil, or mineral oil.
  • the compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
  • compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils.
  • the pharmaceutical composition is applied as a topical ointment or cream.
  • the active ingredient may be employed with either a paraffinic or a water-miscible ointment base.
  • the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
  • Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.
  • Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes. Transmucosal administration may be accomplished through the use of nasal sprays, aerosol sprays, tablets, or suppositories, and transdermal
  • administration may be via ointments, salves, gels, patches, or creams as generally known in the art.
  • a composition comprising the itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II), is encapsulated in a suitable vehicle to either aid in the delivery of the compound to target cells, to increase the stability of the composition, or to minimize potential toxicity of the composition.
  • a suitable vehicle is suitable for delivering a composition of the present invention.
  • suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers, and other phospholipid-containing systems.
  • compositions into delivery vehicles are known in the art.
  • a liposome delivery vehicle may be utilized.
  • Liposomes depending upon the embodiment, are suitable for delivery of the itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II), in view of their structural and chemical properties.
  • liposomes are spherical vesicles with a phospholipid bilayer membrane.
  • the lipid bilayer of a liposome may fuse with other bilayers (e.g., the cell membrane), thus delivering the contents of the liposome to cells.
  • the itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II) may be selectively delivered to a cell by encapsulation in a liposome that fuses with the targeted cell’s membrane.
  • Liposomes may be comprised of a variety of different types of phosolipids having varying hydrocarbon chain lengths.
  • Phospholipids generally comprise two fatty acids linked through glycerol phosphate to one of a variety of polar groups. Suitable phospholids include phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE).
  • PA phosphatidic acid
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • PG phosphatidylglycerol
  • DPG diphosphatidylglycerol
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • the fatty acid chains comprising the phospholipids may range from about 6 to about 26 carbon atoms in length, and the lipid chains may be saturated or unsaturated.
  • Suitable fatty acid chains include (common name presented in parentheses) n-dodecanoate (laurate), n- tretradecanoate (myristate), n-hexadecanoate (palmitate), n-octadecanoate (stearate), n-eicosanoate (arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate), cis-9-hexadecenoate (palmitoleate), cis-9-octadecanoate (oleate), cis,cis-9,12- octadecandienoate (linoleate), all cis-9, 12, 15-octadecatrienoate (linolen
  • phospholipid may be identical or different. Acceptable phospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS, distearoyl PC, dimyristoyl PS, dimyristoyl PC,
  • the phospholipids may come from any natural source, and, as such, may comprise a mixture of phospholipids.
  • egg yolk is rich in PC, PG, and PE
  • soy beans contains PC, PE, PI, and PA
  • animal brain or spinal cord is enriched in PS.
  • Phospholipids may come from synthetic sources too. Mixtures of phospholipids having a varied ratio of individual phospholipids may be used. Mixtures of different phospholipids may result in liposome compositions having advantageous activity or stability of activity properties.
  • phospholipids may be mixed, in optimal ratios with cationic lipids, such as N-(1-(2,3-dioleolyoxy)propyl)-N,N,N- trimethyl ammonium chloride, 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchloarate, 3,3’-deheptyloxacarbocyanine iodide, 1,1’-dedodecyl-3,3,3’,3’- tetramethylindocarbocyanine perchloarate, 1,1’-dioleyl-3,3,3’,3’-tetramethylindo carbocyanine methanesulfonate, N-4-(delinoleylaminostyryl)-N-methylpyridinium iodide, or 1,1,-dilinoleyl-3,3,3’,3’-tetramethylindocarb
  • Liposomes may optionally comprise sphingolipids, in which spingosine is the structural counterpart of glycerol and one of the one fatty acids of a phosphoglyceride, or cholesterol, a major component of animal cell membranes.
  • Liposomes may optionally contain pegylated lipids, which are lipids covalently linked to polymers of polyethylene glycol (PEG). PEGs may range in size from about 500 to about 10,000 daltons.
  • Liposomes may further comprise a suitable solvent.
  • the solvent may be an organic solvent or an inorganic solvent.
  • Suitable solvents include, but are not limited to, dimethylsulfoxide (DMSO), methylpyrrolidone, N-methylpyrrolidone, acetronitrile, alcohols, dimethylformamide, tetrahydrofuran, or combinations thereof.
  • Liposomes carrying the itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II), may be prepared by any known method of preparing liposomes for drug delivery, such as, for example, detailed in U.S. Pat. Nos.4,241,046; 4,394,448; 4,529,561; 4,755,388; 4,828,837; 4,925,661; 4,954,345; 4,957,735; 5,043,164; 5,064,655; 5,077,211; and 5,264,618, the disclosures of which are hereby incorporated by reference in their entirety.
  • liposomes may be prepared by sonicating lipids in an aqueous solution, solvent injection, lipid hydration, reverse evaporation, or freeze drying by repeated freezing and thawing.
  • the liposomes are formed by sonication.
  • the liposomes may be multilamellar, which have many layers like an onion, or unilamellar.
  • the liposomes may be large or small. Continued high-shear sonication tends to form smaller unilamellar lipsomes.
  • all of the parameters that govern liposome formation may be varied. These parameters include, but are not limited to, temperature, pH, concentration of the itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II), concentration and composition of lipid, concentration of multivalent cations, rate of mixing, presence of and concentration of solvent.
  • a composition of the invention may be delivered to a cell as a microemulsion.
  • Microemulsions are generally clear,
  • thermodynamically stable solutions comprising an aqueous solution, a surfactant, and “oil.”
  • the "oil” in this case, is the supercritical fluid phase.
  • the surfactant rests at the oil- water interface.
  • Any of a variety of surfactants are suitable for use in microemulsion formulations including those described herein or otherwise known in the art.
  • the aqueous microdomains suitable for use in the invention generally will have
  • microemulsions can and will have a multitude of different microscopic structures including sphere, rod, or disc shaped aggregates.
  • the structure may be micelles, which are the simplest microemulsion structures that are generally spherical or cylindrical objects. Micelles are like drops of oil in water, and reverse micelles are like drops of water in oil.
  • the microemulsion structure is the lamellae. It comprises consecutive layers of water and oil separated by layers of surfactant. The“oil” of microemulsions optimally comprises phospholipids.
  • any of the phospholipids detailed above for liposomes are suitable for embodiments directed to microemulsions.
  • the compound of the itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II) may be encapsulated in a microemulsion by any method generally known in the art.
  • the itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II), may be delivered in a dendritic macromolecule, or a dendrimer.
  • a dendrimer is a branched tree-like molecule, in which each branch is an interlinked chain of molecules that divides into two new branches (molecules) after a certain length. This branching continues until the branches (molecules) become so densely packed that the canopy forms a globe.
  • the properties of dendrimers are determined by the functional groups at their surface. For example, hydrophilic end groups, such as carboxyl groups, would typically make a water-soluble dendrimer.
  • phospholipids may be incorporated in the surface of a dendrimer to facilitate absorption across the skin. Any of the phospholipids detailed for use in liposome embodiments are suitable for use in dendrimer embodiments. Any method generally known in the art may be utilized to make dendrimers and to encapsulate compositions of the invention therein. For example, dendrimers may be produced by an iterative sequence of reaction steps, in which each additional iteration leads to a higher order dendrimer. Consequently, they have a regular, highly branched 3D structure, with nearly uniform size and shape.
  • the final size of a dendrimer is typically controlled by the number of iterative steps used during synthesis.
  • a variety of dendrimer sizes are suitable for use in the invention.
  • the size of dendrimers may range from about 1 nm to about 100 nm.
  • a safe and effective amount of itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II) is, for example, that amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects.
  • an effective amount of an itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II) described herein can substantially modulate I ⁇ B ⁇ , inhibit an I ⁇ B ⁇ /IL17 associated disease, slow the progress of an I ⁇ B ⁇ /IL17 associated disease, or limit the development of an I ⁇ B ⁇ /IL17 associated disease.
  • a therapeutically effective amount of an itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II) can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient.
  • the compounds of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to modulate I ⁇ B ⁇ , inhibit an I ⁇ B ⁇ /IL17 associated disease, slow the progress of an I ⁇ B ⁇ /IL17 associated disease, or limit the development of an I ⁇ B ⁇ /IL17 associated disease.
  • compositions described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.
  • Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or
  • the dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50, where larger therapeutic indices are generally understood in the art to be optimal.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al.
  • treating a state, disease, disorder, or condition includes preventing or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof.
  • treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms.
  • a benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.
  • Administration of itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II) can occur as a single event or over a time course of treatment.
  • an itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II) can be administered daily, weekly, bi-weekly, or monthly.
  • the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.
  • Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for an I ⁇ B ⁇ /IL17 associated disease.
  • Itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II) can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent.
  • another agent such as an antibiotic, an anti-inflammatory, or another agent.
  • itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II) can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory.
  • Simultaneous administration can occur through administration of separate compositions, each containing one or more of itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II), an antibiotic, an anti-inflammatory, or another agent.
  • Simultaneous administration can occur through administration of one composition containing two or more of itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II), an antibiotic, an anti-inflammatory, or another therapeutic agent for an Ikb- ⁇ associated disease (e.g., chemotherapy, radiation, or immunotherapy for cancer).
  • Itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II) can be administered sequentially with an antibiotic, an anti-inflammatory, or another agent.
  • itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II) can be administered before or after administration of an antibiotic, an anti-inflammatory, or another agent.
  • the present disclosure encompasses a method of treating an I ⁇ B ⁇ associated disease in a subject in need thereof.
  • the method comprises administration of a therapeutically effective amount of itaconate, malonate, derivatives thereof, a compound of Formula (I), or a compound of Formula (II), so as to modulate I ⁇ B ⁇ , inhibit an I ⁇ B ⁇ /IL17 associated disease, slow the progress of an I ⁇ B ⁇ /IL17 associated disease, or limit the development of an I ⁇ B ⁇ /IL17 associated disease.
  • the present disclosure encompasses a method suppressing Ikb- ⁇ induction in a subject in need thereof or in a biological sample, the method comprising administering to the subject, or contacting the biological sample with a composition comprising a therapeutically effective amount a compound of itaconate, malonate, derivatives thereof, a compound of Formula (I), a compound of Formula (II) or combinations thereof.
  • the present disclosure encompasses of inhibiting tumor growth in a subject in need thereof, the method comprising
  • compositions comprising a therapeutically effective amount a compound of itaconate, malonate, derivatives thereof, a compound of Formula (I), a compound of Formula (II) or combinations thereof.
  • the present disclosure provides a composition comprising itaconate, malonate, derivatives thereof, a compound of Formula (I), a compound of Formula (II) or combinations thereof, for use in vitro, in vivo, or ex vivo. Suitable compositions comprising itaconate, malonate, or derivatives thereof are disclosed herein, for instance those described in Section I.
  • a pharmaceutical composition comprising itaconate, malonate, derivatives thereof, a compound of Formula (I), a compound of Formula (II) or combinations thereof can treat, reduce, or prevent a disease, disorder, or condition associated with inflammation or an immune response.
  • diseases associated with inflammation or an immune response can include ischaemia-reperfusion, cardiovascular infarction, inflammatory bowel disease, psoriasis, multiple sclerosis, rheumatoid arthritis auto-inflammatory disease, or an autoimmune disease.
  • the disease, disorder, or condition can be ischaemia-reperfusion in the heart, kidney, or brain or a tissue injury caused by ischaemia-reperfusion in the heart, kidney, or brain, or myocardial injury, where the tissue injury can occur during reperfusion.
  • compositions of the disclosure have been shown to modulate the expression or secretion of Casp1, iNOS, HIF-1 ⁇ , pro-IL-1 ⁇ , ASC, NLRP3, NOS2, iNOS, IL6, IL12B, IFNB1, IL-12p70, IL-6, IL-1 ⁇ , IL-12 ⁇ , NO, GM-CSF, IL-17, or IL-18.
  • compositions as described herein can treat a disease, disorder, or condition associated with increased expression or secretion of Casp1, iNOS, HIF-1 ⁇ , pro-IL-1 ⁇ , ASC, NLRP3, NOS2, iNOS, IL6, IL12B, IFNB1, IL-12p70, IL-6, IL-1 ⁇ , IL-12 ⁇ , NO, GM-CSF, IL-17, or IL-18.
  • Diseases, disorders, and conditions that can be treated by the compounds of the disclosure include: adult and juvenile Still disease; asthma; allergy; Alzheimer’s disease; age-related macular degeneration; antisynthetase syndrome; autoinflammatory disease; autoimmune disease; autoimmune response; Behçet disease; Blau syndrome; cancer; cardiovascular infarction; chronic infantile neurological cutaneous and articular (CINCA) syndrome; chronic recurrent multifocal osteomyelitis; cinca syndrome; classic autoinflammatory diseases; cryopyrin-associated
  • CAS autoinflammatory syndromes
  • DIRA deficiency in IL-1 receptor antagonist
  • diabetes mellitus a malignant neoplasm originating from the central nervous system
  • Erdheim-Chester syndrome a malignant neoplasm originating from the central nervous system
  • tuberculosis tuberculosis; familial atypical mycobacteriosis; familial cold autoinflammatory syndrome (FCAS); gastric cancer Risk after H. pylori Infection; Guillain–Barré syndrome;
  • FCAS familial cold autoinflammatory syndrome
  • RA rheumatoid arthritis
  • Sapho Syndrome rheumatoid arthritis
  • Schnitzler syndrome secondary rheumatoid arthritis
  • Cytokines are considered to be in a broad and loose category of small proteins ( ⁇ 5–20 kDa) that are important in cell signaling. Their release has an effect on the behavior of cells around them. It can be said that cytokines are involved in autocrine signaling, paracrine signaling and endocrine signaling as immunomodulating agents. Their definite distinction from hormones is still part of ongoing research.
  • Cytokines are generally known to include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors but generally not hormones or growth factors (despite some overlap in the terminology). Cytokines can be produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells; a given cytokine may be produced by more than one type of cell.
  • Cytokines can act through receptors, and are especially important in the immune system. Cytokines can modulate the balance between humoral and cell- based immune responses, and they can regulate the maturation, growth, or
  • cytokines can enhance or inhibit the action of other cytokines in complex ways.
  • Cytokines are different from hormones, which can also be important in cell signaling molecules, in that hormones circulate in less variable concentrations and hormones tend to be made by specific kinds of cells.
  • Cytokines can be important in health and disease, specifically in host responses to infection, immune responses, inflammation, trauma, sepsis, cancer, or reproduction.
  • compositions as described herein can treat a disease, disorder, or condition associated with increased expression or secretion of Casp1, iNOS, HIF-1 ⁇ , pro-IL-1 ⁇ , ASC, NLRP3, NF-kappa-B, NOS2, iNOS, IL6, IL12B, IFNB1, IL-12p70, IL-6, IL-1 ⁇ , IL-12 ⁇ , NO, GM-CSF, IL-17, or IL-18.
  • Ikb- ⁇ /IL17 and NF- ⁇ associated diseases a disease, disorder, or condition associated with increased expression or secretion of Casp1, iNOS, HIF-1 ⁇ , pro-IL-1 ⁇ , ASC, NLRP3, NF-kappa-B, NOS2, iNOS, IL6, IL12B, IFNB1, IL-12p70, IL-6, IL-1 ⁇ , IL-12 ⁇ , NO, GM-CSF, IL-17, or IL-18.
  • Interleukin-17 (IL-17) family cytokines have recently emerged as important players in inflammatory responses.
  • IL-17A and IL-17F which are most highly related among IL-17 family cytokines, are expressed by a distinct T cell subset, Th17 cells.
  • I ⁇ B ⁇ was identified to be induced by STAT3 and promote Th17 cell differentiation.
  • NF-kappa-B inhibitor zeta is a protein that in humans is encoded by the NFKBIZ gene.
  • This gene is a member of the Ankyrin-repeat family and is induced by lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • NF- ⁇ B associated inflammatory diseases can include rheumatoid arthritis (RA), atherosclerosis, multiple sclerosis, chronic inflammatory demyelinating polyradiculoneuritis, asthma, inflammatory bowel disease, heliobacter pylori-associated gastritis, or systemic inflammatory response syndrome (Tak et al. J Clin Invest.2001 Jan 1; 107(1): 7–11).
  • RA rheumatoid arthritis
  • atherosclerosis multiple sclerosis
  • chronic inflammatory demyelinating polyradiculoneuritis asthma
  • inflammatory bowel disease inflammatory bowel disease
  • heliobacter pylori-associated gastritis or systemic inflammatory response syndrome
  • NF- ⁇ B associated cancer can include prostate cancer, breast cancer, lung cancer, head and neck squamous cell carcinomas, glioblastoma, skin cancer, brain cancer, glioma, liver cancer, non-small cell lung cancers (NSCLC), lymphoma, leukemia, rectal cancer, gastric cancer, or colon cancer (Xia et al. Cancer Immunol Res.2014 Sep; 2(9): 823–830).
  • NSCLC non-small cell lung cancers
  • An Ikb ⁇ /IL17 associated disease can be psoriasis, multiple sclerosis, or activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), but not to GCB subtype.
  • ABC DLBCL is associated with substantially worse outcomes when treated with standard chemoimmunotherapy.
  • the ABC subtype is characterized by chronic active B-cell receptor (BCR) signaling, which stimulates NF- ⁇ B activity.
  • Ikb ⁇ /IL17 associated diseases can be a Ikb ⁇ /IL17 associated carcinoma, an autoimmune disease, Sjögren's syndrome (or a Sjögren's syndrome-like autoimmune disease), lupus, myxoid liposarcoma, brain glioblastoma multiforme, hypersensitivity syndrome, multiple sclerosis, susceptibility to pneumococcal disease, ocular surface inflammatory disorders, epilepsy, or meningococcal meningitis, atopic dermatitis, bursitis, tendinitis, psoriasis, or allergic conjunctivitis.
  • Sjögren's syndrome or a Sjögren's syndrome-like autoimmune disease
  • lupus myxoid liposarcoma
  • brain glioblastoma multiforme glioblastoma multiforme
  • hypersensitivity syndrome multiple sclerosis
  • susceptibility to pneumococcal disease ocular surface inflammatory disorders
  • Interleukin 17 is a pro-inflammatory cytokine produced by T-helper cells, gamma-delta T cells and subsets of innate lymphoid cells (Sutton et al, EJI 2012; Klose and Artis, Nat Immunol, 2016), and is induced and/or promoted by cytokines including IL-6, IL-23, IL-1 ⁇ , or TGF ⁇ .
  • cytokines including IL-6, IL-23, IL-1 ⁇ , or TGF ⁇ .
  • IL-17R type I cell surface receptor of which there are at least three variants IL17RA, IL17RB, and IL17RC.
  • IL-17 acts as a potent mediator in delayed-type reactions by increasing chemokine production in various tissues.
  • IL-17 Signaling from IL-17 recruits monocytes and neutrophils to the site of inflammation in response to invasion by pathogens, similar to Interferon gamma.
  • IL-17 has been demonstrated to act synergistically with tumor necrosis factor and interleukin-1. This activity can also be redirected towards the host and result in various autoimmune disorders that involve chronic inflammation, such as the skin disorder psoriasis.
  • IL-17 is implicated in numerous inflammatory diseases (see e.g., psoriasis, vitiligo, allergies, autoimmune disease, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, or asthma) (Wang et al PlosOne 2011).
  • IL-17 has also been associated with cancer.
  • an IL-17 associated cancer can be breast cancer, lung cancer, colorectal cancer (CRC), prostate cancer, breast cancer, myeloma, melanoma, ovarian cancer, renal cell carcinoma, colon cancer, acute myeloid leukemia, gastric cancer, lymphoma, pancreatic cancer, or lung cancer (Murugaiyan et al. J Immunol 2009; 183:4169-4175)
  • Ikb- ⁇ is an inducible nuclear protein that regulates Toll/IL-1- receptor-mediated gene expression and has been shown to be critical for production of IL6 but not TNF ⁇ in macrophages4.
  • Ikb- ⁇ is an inducible nuclear protein that regulates Toll/IL-1- receptor-mediated gene expression and has been shown to be critical for production of IL6 but not TNF ⁇ in macrophages4.
  • the inventors have also shown, itaconate and derivatives thereof can down-regulate IL17-induced Ikb ⁇ activation.
  • itaconate and derivatives thereof have been shown to inhibit STAT3 activation. DI inhibits STAT3 activation in macrophages by mechanism that does not involve Nrf2.
  • Interleukin 1 beta is a cytokine protein that in humans is encoded by the IL1B gene.
  • IL1B gene There are two genes for interleukin-1 (IL-1): IL-1 alpha and IL-1 beta.
  • IL-1 ⁇ precursor is cleaved by cytosolic caspase 1 (interleukin 1 beta convertase) to form mature IL-1 ⁇ .
  • Increased production of IL-1 ⁇ can causes a number of different autoinflammatory syndromes, most notably the monogenic conditions referred to as Cryopyrin-Associated Autoinflammatory Syndromes (CAPS), due to mutations in the inflammasome receptor NLRP3 which triggers processing of IL-1 ⁇ .
  • Cryopyrin-Associated Autoinflammatory Syndromes Cryopyrin-Associated Autoinflammatory Syndromes
  • IL-1 ⁇ can be associated with a number of autoinflammatory diseases. For these, neutralization of IL-1 ⁇ results in a rapid and sustained reduction in disease severity. Treatment for autoimmune diseases often includes
  • IL-1 ⁇ implicated diseases can include gout, type 2 diabetes, heart failure, recurrent pericarditis, rheumatoid arthritis, and smoldering myeloma also are responsive to IL-1 ⁇ neutralization.
  • inflammatory diseases see e.g., Dinarello, Blood.2011 Apr 7; 117(14): 3720–3732.
  • Classic autoinflammatory diseases Familial Mediterranean fever (FMF); Pyogenic arthritis, pyoderma gangrenosum, acne (PAPA); Cryopyrin-associated periodic syndromes (CAPS); Hyper IgD syndrome (HIDS); Adult and juvenile Still disease;
  • TRAPS TNF receptor-associated periodic syndrome
  • Blau syndrome Blau syndrome
  • Sweet syndrome Deficiency in IL-1 receptor antagonist (DIRA)
  • DIRA Recurrent idiopathic pericarditis
  • Macrophage activation syndrome MAS
  • Urticarial vasculitis Antisynthetase syndrome; Relapsing chondritis; Behçet disease; Erdheim-Chester syndrome (histiocytosis) ; Synovitis, acne, pustulosis, hyperostosis, osteitis (SAPHO); Rheumatoid arthritis; Periodic fever, aphthous stomatitis, pharyngitis, adenitis syndrome (PFAPA) ; Urate crystal arthritis (gout); Type 2 diabetes; Smoldering multiple myeloma; Postmyocardial infarction heart failure; or Osteoarthritis.
  • TRAPS TNF receptor-associated periodic syndrome
  • DIRA Recurrent
  • Interleukin 17 is a pro-inflammatory cytokine produced by T-helper cells, gamma-delta T cells and subsets of innate lymphoid cells (Sutton et al, EJI 2012; Klose and Artis, Nat Immunol, 2016), and is induced and/or promoted by cytokines including IL-6, IL-23, IL-1 ⁇ , or TGF ⁇ .
  • cytokines including IL-6, IL-23, IL-1 ⁇ , or TGF ⁇ .
  • IL-17R type I cell surface receptor of which there are at least three variants IL17RA, IL17RB, and IL17RC.
  • IL-17 acts as a potent mediator in delayed-type reactions by increasing chemokine production in various tissues.
  • IL-17 Signaling from IL-17 recruits monocytes and neutrophils to the site of inflammation in response to invasion by pathogens, similar to Interferon gamma.
  • IL-17 has been demonstrated to act synergistically with tumor necrosis factor and interleukin-1. This activity can also be redirected towards the host and result in various autoimmune disorders that involve chronic inflammation, such as the skin disorder psoriasis.
  • IL-17 is implicated in numerous inflammatory diseases (see e.g., psoriasis, vitiligo, allergies, autoimmune disease, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, or asthma) (Wang et al PlosOne 2011).
  • IL-18 has been shown to induce severe inflammatory reactions, which suggests its role in certain inflammatory disorders.
  • IL-18 has been implicated in age-related macular degeneration, Hashimoto's thyroiditis, Alzheimer’s disease.
  • Casp1 associated diseases (e) Casp1 associated diseases [00129] Caspase-1/Interleukin-1 converting enzyme (ICE) plays a central role in cell immunity as an inflammatory response initiator. Caspase-1 has also been shown to induce necrosis and may also function in various developmental stages.
  • ICE Interleukin-1 converting enzyme
  • Nitric oxide synthases are a family of enzymes catalyzing the production of nitric oxide (NO) from L-arginine. NO is an important cellular signaling molecule. It helps modulate vascular tone, insulin secretion, airway tone, and
  • Nitric oxide is mediated in mammals by the calcium- calmodulin controlled isoenzymes eNOS (endothelial NOS) and nNOS (neuronal NOS).
  • eNOS endothelial NOS
  • nNOS neuroneuronal NOS
  • the inducible isoform, iNOS is involved in immune response, binds calmodulin at physiologically relevant concentrations, and produces NO as an immune defense mechanism, as NO is a free radical with an unpaired electron. It is the proximate cause of septic shock and may function in autoimmune disease.
  • Hypoxia-inducible factor 1-alpha also known as HIF-1-alpha
  • HIF-1-alpha is a subunit of a heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1) that is encoded by the HIF1A gene. It is a basic helix-loop-helix PAS domain containing protein, and is considered as the master transcriptional regulator of cellular and developmental response to hypoxia.
  • the dysregulation and overexpression of HIF1A by either hypoxia or genetic alternations have been heavily implicated in cancer biology, as well as a number of other pathophysiologies, specifically in areas of vascularization and angiogenesis, energy metabolism, cell survival, and tumor invasion. Two other alternative transcripts encoding different isoforms have been identified.
  • Apoptosis-associated speck-like protein containing a CARD or ASC is a protein that in humans is encoded by the PYCARD gene.
  • This gene encodes an adaptor protein that is composed of two protein–protein interaction domains: an N-terminal PYRIN-PAAD-DAPIN domain (PYD) and a C-terminal caspase-recruitment domain (CARD).
  • PYD and CARD domains are members of the six-helix bundle death domain-fold superfamily that mediates assembly of large signaling complexes in the inflammatory and apoptotic signaling pathways via the activation of caspase.
  • this protein In normal cells, this protein is localized to the cytoplasm; however, in cells undergoing apoptosis, it forms ball-like aggregates near the nuclear periphery. Two transcript variants encoding different isoforms have been found for this gene.
  • PYCARD Diseases associated with PYCARD include Chronic Recurrent Multifocal Osteomyelitis and Cinca Syndrome.
  • NACHT, LRR and PYD domains-containing protein 3 (NALP3) also known by cryopyrin is a protein that in humans is encoded by the NLRP3 gene located on the long arm of chromosome 1.
  • NALP3 is expressed predominantly in macrophages and as a component of the inflammasome, detects products of damaged cells such as
  • NALP3 extracellular ATP and crystalline uric acid.
  • Activated NALP3 in turn triggers an immune response. Mutations in the NLRP3 gene are associated with a number of organ specific autoimmune diseases.
  • cryopyrin-associated periodic syndrome This includes familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurological cutaneous and articular (CINCA) syndrome, and neonatal-onset multisystem inflammatory disease (NOMID).
  • FCAS familial cold autoinflammatory syndrome
  • MFS Muckle-Wells syndrome
  • CINCA chronic infantile neurological cutaneous and articular
  • NOMID neonatal-onset multisystem inflammatory disease
  • NALP3 inflammasome has a role in the pathogenesis of gout and neuroinflammation occurring in protein-misfolding diseases, such as Alzheimer's, Parkinson's, and Prion diseases.
  • NALP3 has been connected with carcinogenesis. For example, all the components of the NALP3 inflammasome are downregulated or completely lost in human hepatocellular carcinoma.
  • NALP3 Diseases associated with NALP3 are familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurological cutaneous and articular (CINCA) syndrome, and neonatal-onset multisystem
  • NOMID inflammatory disease
  • Interleukin 6 is an interleukin that acts as both a pro- inflammatory cytokine and an anti-inflammatory myokine. In humans, it is encoded by the IL6 gene.
  • Interleukin 6 is secreted by B cells, T cells, and macrophages to stimulate immune response, e.g. during infection and after trauma, especially burns or other tissue damage leading to inflammation.
  • IL-6 also plays a role in fighting infection, as IL-6 has been shown in mice to be required for resistance against bacterium
  • osteoblasts secrete IL-6 to stimulate osteoclast formation.
  • Smooth muscle cells in the tunica media of many blood vessels also produce IL-6 as a pro-inflammatory cytokine.
  • IL-6's role as an anti-inflammatory cytokine is mediated through its inhibitory effects on TNF-alpha and IL-1, and activation of IL-1ra and IL-10.
  • IL-6 is associated with and stimulates the inflammatory and auto- immune processes in many diseases such as diabetes, atherosclerosis, depression, Alzheimer's Disease, systemic lupus erythematosus, multiple myeloma, prostate cancer, Behçet's disease, inflammatory bowel disease (Neurath, Nat Rev Immunol, 2014), rheumatoid arthritis, vitiligo, and systemic sclerosis (O’Reilly et al, Clin and Translational Immunol, 2013).
  • IL-6 has been associated with diabetes mellitus and systemic juvenile rheumatoid arthritis.
  • Interferons are a group of signaling proteins made and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses.
  • IFNs belong to the large class of proteins known as cytokines, molecules used for communication between cells to trigger the protective defenses of the immune system that help eradicate pathogens. Interferons are named for their ability to "interfere” with viral replication by protecting cells from virus infections. IFNs also have various other functions: they activate immune cells, such as natural killer cells and macrophages; they increase host defenses by up-regulating antigen presentation by virtue of increasing the expression of major histocompatibility complex (MHC) antigens. Certain symptoms of infections, such as fever, muscle pain and "flu-like symptoms", are also caused by the production of IFNs and other cytokines.
  • MHC major histocompatibility complex
  • Interferon beta is a protein that in humans is encoded by the IFNB1 gene.
  • Diseases associated with IFNB1 include Relapsing-Remitting Multiple Sclerosis and Secondary Progressive Multiple Sclerosis.
  • Subunit beta of interleukin 12 is a protein that in humans is encoded by the IL12B gene.
  • IL-12B is a common subunit of interleukin 12 and Interleukin 23.
  • This gene encodes a subunit of interleukin 12, a cytokine that acts on T and natural killer cells, and has a broad array of biological activities.
  • Interleukin 12 is a disulfide-linked heterodimer composed of the 40 kD cytokine receptor like subunit encoded by this gene, and a 35 kD subunit encoded by IL12A.
  • This cytokine is expressed by activated macrophages that serve as an essential inducer of Th1 cells development. This cytokine has been found to be important for sustaining a sufficient number of memory/effector Th1 cells to mediate long-term protection to an intracellular pathogen. Overexpression of this gene was observed in the central nervous system of patients with multiple sclerosis (MS), suggesting a role of this cytokine in the
  • the promoter gene polymorphism of this gene has been reported to be associated with the severity of atopic and non-atopic asthma in children.
  • Interleukin 12 is an interleukin that is naturally produced by dendritic cells, macrophages, neutrophils, and human B-lymphoblastoid cells (NC-37) in response to antigenic stimulation.
  • IL-12 is composed of a bundle of four alpha helices. It is a heterodimeric cytokine encoded by two separate genes, IL-12A (p35) and IL-12B (p40).
  • the active heterodimer (referred to as 'p70'), and a homodimer of p40 are formed following protein synthesis.
  • IL-12 is linked with autoimmunity.
  • Administration of IL-12 to people suffering from autoimmune diseases was shown to worsen the autoimmune
  • IL-12 gene knock-out in mice or a treatment of mice with IL-12 specific antibodies ameliorated the disease.
  • Interleukin 12 is produced by activated antigen-presenting cells (dendritic cells, macrophages). It promotes the development of Th1 responses and is a powerful inducer of IFN ⁇ production by T and NK cells.
  • IL12B diseases associated with IL12B include Immunodeficiency 29, Mycobacteriosis and Familial Atypical Mycobacteriosis.
  • IL-12p70 has been shown to be overexpressed in Crohn’s disease.
  • Dysregulated expression of IL-12 p40 can lead to prolonged, unresolved inflammation manifesting into chronic inflammatory disorders such as inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • IL12RB1 Diseases associated with IL12RB1 include Immunodeficiency 30 and Familial Atypical Mycobacteriosis.
  • m GM-CSF associated diseases
  • Granulocyte-macrophage colony-stimulating factor also known as colony stimulating factor 2 (CSF2)
  • CSF2 colony stimulating factor 2
  • GM-CSF is a monomeric glycoprotein secreted by macrophages, T cells, B cells, mast cells, NK cells, endothelial cells and fibroblasts that functions as a cytokine.
  • the pharmaceutical analogs of naturally occurring GM-CSF are called sargramostim and molgramostim.
  • GM-CSF is found in high levels in joints with rheumatoid arthritis, in the cerebrospinal fluid of MS patients and in the serum of patients with acute aortic aneurysm. Also, its receptor is highly expressed in subsets of myeloid cells in patients with rheumatoid arthritis and psoriatic arthritis. GM-CSF can activate microglial cells that promote inflammation of the central nervous system. Targeting GM-CSF may reduce inflammation or damage and could be beneficial for patients with rheumatoid arthritis, MS, plaque psoriasis, and asthma (Wicks and Roberts, Nat Rev Rheumatology, 2016).
  • P2X purinoceptor 7 is a protein that in humans is encoded by the P2RX7 gene.
  • the product of this gene belongs to the family of purinoceptors for ATP. Multiple alternatively spliced variants which would encode different isoforms have been identified although some fit nonsense-mediated decay criteria.
  • the receptor is found in the central and peripheral nervous systems, in microglia, in macrophages, in uterine endometrium, and in the retina.
  • the P2X7 receptor also serves as a pattern recognition receptor for extracellular ATP- mediated apoptotic cell death, regulation of receptor trafficking, mast cell degranulation, and inflammation.
  • Microglial P2X7 receptors are thought to be involved in neuropathic pain because blockade or deletion of P2X7 receptors results in decreased responses to pain, as demonstrated in vivo.
  • P2X7 receptor signaling increases the release of pro-inflammatory molecules such as IL-1 ⁇ , IL-6, and TNF- ⁇ .
  • P2X7 receptors have been linked to increases in pro-inflammatory cytokines such as CXCL2 and CCL3.
  • P2X7 receptors are also linked to P2X4 receptors, which are also associated with neuropathic pain mediated by microglia.
  • P2RX7 has also been linked to osteoporosis. Mutations in this gene have been associated to low lumbar spine bone mineral density and accelerated bone loss in post-menopausal women.
  • P2RX7 has also been linked to diabetes.
  • the ATP/P2X7R pathway may trigger T-cell attacks on the pancreas, rendering it unable to produce insulin.
  • This autoimmune response may be an early mechanism by which the onset of diabetes is caused.
  • P2RX7 has also been linked to hepatic fibrosis.
  • One study in mice showed that blockade of P2X7 receptors attenuates onset of liver fibrosis.
  • LPS lipopolysaccharide
  • immunomodulatory agents can include: autoimmune disease and responses, MS flare ups, Guillain–Barré syndrome and a variant of Guillain–Barré called Miller-Fisher syndrome.
  • a subject may be a rodent, a human, a livestock animal, a companion animal, or a zoological animal.
  • the subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc.
  • the subject may be a livestock animal.
  • suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas and alpacas.
  • the subject may be a companion animal.
  • companion animals may include pets such as dogs, cats, rabbits, and birds.
  • the subject may be a zoological animal.
  • a“zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In a preferred embodiment, the subject is a human. III. KITS
  • kits can include an agent or
  • composition described herein and, in certain embodiments, instructions for
  • kits can facilitate performance of the methods described herein.
  • the different components of the composition can be packaged in separate containers and admixed immediately before use.
  • Components include, but are not limited to compositions and pharmaceutical formulations comprising an Ikb- ⁇ modulation agent.
  • Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition.
  • the pack may, for example, comprise metal or plastic foil such as a blister pack.
  • Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.
  • Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately.
  • sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen.
  • Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents.
  • suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy.
  • Other containers include test tubes, vials, flasks, bottles, syringes, and the like.
  • Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle.
  • Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix.
  • Removable membranes may be glass, plastic, rubber, and the like.
  • kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, mini-CD-ROM, CD- ROM, DVD-ROM, Zip disc, videotape, audio tape, and the like. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.
  • compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P.1988.
  • alkyl refers to saturated monovalent hydrocarbon radicals having straight or branched hydrocarbon chains or, in the event that at least 3 carbon atoms are present, cyclic hydrocarbons or combinations thereof and contains 1 to 20 carbon atoms (C.sub.1-20alkyl), suitably 1 to 10 carbon atoms (C.sub.1-10alkyl), preferably 1 to 8 carbon atoms (C.sub.1-8alkyl), more preferably 1 to 6 carbon atoms (C.sub.1-4alkyl), and even more preferably 1 to 4 carbon atoms (C.sub.1-4alkyl).
  • alkyl radicals include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
  • alkenyl refers to monovalent hydrocarbon radicals having a straight or branched hydrocarbon chains having one or more double bonds and containing from 2 to about 18 carbon atoms, preferably from 2 to about 8 carbon atoms, more preferably from 2 to about 5 carbon atoms.
  • suitable alkenyl radicals include ethenyl, propenyl, alkyl, 1,4- butadienyl, and the like.
  • alkynyl refers to monovalent hydrocarbon radicals having a straight or branched hydrocarbon chains having one or more triple bonds and containing from 2 to about 10 carbon atoms, more preferably from 2 to about 5 carbon atoms.
  • alkynyl radicals include ethynyl, propynyl, (propargyl), butyny,l and the like.
  • aryl as used herein, alone or as part of a group, includes an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, and includes monocyclic and polycyclic radicals, such as phenyl, biphenyl, naphthyl.
  • alkoxy refers to an alkyl ether radical wherein the term alkyl is as defined above.
  • alkyl ether radical include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, and the like.
  • cycloalkyl as used herein, alone or in combination, means a saturated or partially saturated monocyclic, bicyclic or tricyclic alkyl radical wherein each cyclic moiety contains from about 3 to about 8 carbon atoms, more preferably from about 3 to about 6 carbon atoms.
  • examples of such cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
  • cycloalkylalkyl as used herein, alone or in combination, means an alkyl radical as defined above which is substituted by a cycloalkyl radical as defined above.
  • examples of such cycloalkylalkyl radicals include cyclopropylmethyl, cyclobutyl-methyl, cyclopentylmethyl, cyclohexylmethyl, 1-cyclopentylethyl, 1- cyclohexylethyl, 2-cyclopentylethyl, 2-cyclohexylethyl, cyclobutylpropyl,
  • substituted means that one or more of the hydrogen atoms bonded to carbon atoms in the chain or ring have been replaced with other substituents.
  • Suitable substituents include monovalent hydrocarbon groups including alkyl groups such as methyl groups and monovalent heterogeneous groups including alkoxy groups such as methoxy groups.
  • branched as used herein means that the carbon chain is not simply a linear chain. "Unbranched” means that the carbon chain is a linear carbon chain.
  • saturated means that the carbon chain or ring does not contain any double or triple bonds.
  • Unsaturated means that the carbon chain or ring contains at least one double bond.
  • An unsaturated carbon chain or ring may include more than one double bond.
  • hydrocarbon group means a chain of 1 to 25 carbon atoms, suitably 1 to 12 carbon atoms, more suitably 1 to 10 carbon atoms, and most suitably 1 to 8 carbon atoms. Hydrocarbon groups may have a linear or branched chain structure. Suitably the hydrocarbon groups have one branch.
  • Carbocyclic group means a saturated or unsaturated hydrocarbon ring.
  • Carbocyclic groups are not aromatic.
  • Carbocyclic groups are monocyclic or polycyclic.
  • Polycyclic carbocyclic groups can be fused, spiro, or bridged ring systems.
  • Monocyclic carbocyclic groups contain 4 to 10 carbon atoms, suitably 4 to 7 carbon atoms, and more suitably 5 to 6 carbon atoms in the ring.
  • Bicyclic carbocyclic groups contain 8 to 12 carbon atoms, preferably 9 to 10 carbon atoms in the rings.
  • heteroatom means an atom other than carbon e.g., in the ring of a heterocyclic group or the chain of a heterogeneous group.
  • heteroatoms are selected from the group consisting of sulfur, phosphorous, nitrogen and oxygen atoms. Groups containing more than one heteroatom may contain different heteroatoms.
  • heterocyclic group means a saturated or unsaturated ring structure containing carbon atoms and 1 or more heteroatoms in the ring.
  • Heterocyclic groups are not aromatic. Heterocyclic groups are monocyclic or polycyclic. Polycyclic heteroaromatic groups can be fused, spiro, or bridged ring systems.
  • Monocyclic heterocyclic groups contain 4 to 10 member atoms (i.e., including both carbon atoms and at least 1 heteroatom), suitably 4 to 7, and more suitably 5 to 6 in the ring.
  • Bicyclic heterocyclic groups contain 8 to 18 member atoms, suitably 9 or 10 in the rings.
  • solvate is intended to mean a solvate form of a specified compound that retains the effectiveness of such compound.
  • solvates include compounds of the invention in combination with, for example: water,
  • DMSO dimethylsulfoxide
  • the term“mmol”, as used herein, is intended to mean millimole.
  • the term“equiv”, as used herein, is intended to mean equivalent.
  • the term“mL”, as used herein, is intended to mean milliliter.
  • the term“g”, as used herein, is intended to mean gram.
  • the term“kg”, as used herein, is intended to mean kilogram.
  • the term“ ⁇ g”, as used herein, is intended to mean micrograms.
  • the term“h”, as used herein, is intended to mean hour.
  • the term“min”, as used herein, is intended to mean minute.
  • the term“M”, as used herein, is intended to mean molar.
  • the term “ ⁇ L”, as used herein, is intended to mean microliter.
  • the term“ ⁇ M”, as used herein, is intended to mean micromolar.
  • the term“nM”, as used herein, is intended to mean nanomolar.
  • the term“N”, as used herein, is intended to mean normal.
  • the term“amu”, as used herein, is intended to mean atomic mass unit.
  • the term“°C”, as used herein, is intended to mean degree Celsius.
  • the term“wt/wt”, as used herein, is intended to mean
  • the term“v/v”, as used herein, is intended to mean volume/volume.
  • the term“MS”, as used herein, is intended to mean mass spectroscopy.
  • the term“HPLC”, as used herein, is intended to mean high performance liquid chromatograph.
  • the term “RT”, as used herein, is intended to mean room temperature.
  • the term “e.g.”, as used herein, is intended to mean example.
  • the term“N/A”, as used herein, is intended to mean not tested.
  • the expression“pharmaceutically acceptable salt” refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention.
  • Preferred salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,
  • a pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion.
  • the counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound.
  • a pharmaceutically acceptable salt may have more than one charged atom in its structure.
  • a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion.
  • the expression“pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules and a compound of the invention.
  • solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
  • the expression“pharmaceutically acceptable hydrate” refers to a compound of the invention, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.
  • diastereoisomers may have a syn- or anti-configuration
  • substituents on bivalent cyclic saturated radicals may have either the cis- or trans-configuration
  • alkenyl radicals may have the E or Z-configuration. All stereochemically isomeric forms of said
  • transition term“consisting of” excludes any element, step, or ingredient not specified in the claims.
  • the transition term“consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.
  • DI Dimethyl itaconate
  • Nrf2-triggering agents modulate inflammatory conditions by down-regulating nuclear factor-k B (NFkB) pathway
  • DI did not show effect on classical NFkB signaling as shown by activation of key NFkB regulators and p65 nuclear localization in LPS-activated cells. Therefore its selective action towards NFkB primary response genes was tested.
  • DI treatment selectively down-regulates Ikb- ⁇ induction in an Nrf2-independent manner.
  • DI showed to be potent inhibitor of Ikb- ⁇ in IL-17A- stimulated keratinocytes where Ikb- ⁇ plays a key role in induction of psoriasis- associated genes. Moreover, DI was capable to ameliorate psoriatic pathology associated in murine model of psoriasis. Similarly, DI was able to ameliorate clinical development of EAE disease. Finally, since Ikb- ⁇ has been also shown to control survival of ABC but not GCB DLBCL cell lines, DI potential to limit proliferation of DLBCL cell lines was tested. DI was selectively toxic to ABC but not GCB subtypes of DLBCL cell lines.
  • DI can be used as a therapeutic option for treatment of Ikbz/Il17 associated diseases such as 1) autoinflammatory diseases (e.g., psoriasis and multiple sclerosis) or 2) Ikbz-dependent tumors such as ABC subtype of DLBCL.
  • autoinflammatory diseases e.g., psoriasis and multiple sclerosis
  • Ikbz-dependent tumors such as ABC subtype of DLBCL.
  • DI-up-regulated genes in mouse macrophages by global transcriptomic profiling.
  • Analysis of DI-pretreated cells identified genes significantly enriched by DI treatment as several prototypical oxidative stress response genes that are commonly up-regulated in cells exposed to oxidative and xenobiotic/electrophilic stress (see e.g., FIG.1A).
  • DI is a methyl ester of itaconic acid, or methylenesuccinic acid that poses electrophilic unsaturated bond which can undergo Michael additions with nucleophiles as SH groups in cysteine (see e.g., FIG.1B).
  • Nfr2/Keap1 antioxidant system is a master regulator of cellular response to electrophilic stress.
  • a cysteine-rich protein Keap1 acts as a redox sensor and upon addition/oxidation of its sulfhydryl groups activates Nrf2 transcription factor.12 h pretreatment of macrophages with DI triggered strong Nrf2 response as detected by Nrf2 western blot and induction of Nrf2 target genes (see e.g., FIG.1C).
  • DI-treated cells In line with upregulation of oxidative signature pathways DI-treated cells exhibited increased intracellular ROS (see e.g., FIG.1D) production and substantial drop in cellular GSH (see e.g., FIG.1E) and GSH/GSSG ratio (see e.g., FIG.1F).
  • DI e.g., DMF, DEM
  • canonical NFkB response was not affected by DI in concentrations effective to inhibit IL6 production but not TNF as detected by p65 nuclear translocation (see e.g., FIG.2A), IKK activation (see e.g., FIG.2B), and IkBa degradation (see e.g., FIG.2C)
  • possible NFkB target genes with selective function in cytokine response was explored.
  • Ikb- ⁇ is an inducible nuclear protein that regulates Toll/IL-1-receptor-mediated gene expression and has been shown to be critical for production of IL6 but not TNFa in macrophages.
  • LPS induced strong expression of Ikb- ⁇ as detected on mRNA and protein level and DI pretreatment resulted in almost complete inhibition of Ikb- ⁇ induction of protein level (see e.g., FIG.2D) with only moderate effect on mRNA level (see e.g., FIG.2E).
  • NAC pretreatment In agreement with neutralizing effect of NAC on downregulation of cytokine production by DI, NAC pretreatment also restored Ikb- ⁇ production in DI-pretreated cells (see e.g., FIG.2F).
  • Ikb- ⁇ protein levels were not due to proteasomal degradation since inhibition of proteasome with MG132 did not affect Ikb- ⁇ levels in DI treated cells (see e.g., FIG.2G). These data suggest that DI acts on NFkBiz mRNA expression and/or interferes with posttranscriptional regulation of Ikb- ⁇ . Ikb- ⁇ expression in LPS-stimulated human monocytes was also tested. DI treatment of monocytes resulted in decreased Ikb- ⁇ protein levels (see e.g., FIG.2H) and inhibition of cytokine production (see e.g., FIG.2I).
  • Ikb- ⁇ mRNA has been previously shown to be regulated by several mechanisms affecting mRNA 3-UTR elements. Therefore Ikb- ⁇ 3-UTR reporter system was utilized in mouse BV2 immortalized cell line. DI pretreatment and LPS stimulation did not affect expression of reporter gene GFP as detected by flow cytometry (see e.g., FIG.2J). Ikb- ⁇ mRNA has been shown to be negatively regulated by Regnase-1. Upon various cell stimuli (T cells, macrophages), this regulation is relieved by Regnase-1 degradation by MALT-1. DI treatment up-regulated Regnase-1 in unstimulated macrophages that was subsequently degraded upon LPS stimulation to a similar extent as in control cells (see e.g., FIG.2K).
  • DI acts on Ikb- ⁇ induction in NRF2-independent manner
  • DI acts on STAT3 activation in NRF2-independent manner
  • Inhibition of STAT3 in macrophages leads to down-regulation of IL6 but not TNF ⁇ production. It was tested whether beside specific NFkB components DI could have additional effect of STAT3 activation.
  • STAT3 was activated upon 1 h and 4 h of LPS stimulation as detected by Y705 site phosphorylation (see e.g., FIG.3B).
  • Nrf2 KO showed no differences in extent of inhibition of STAT3 activation compared with WT cells.
  • DI inhibits STAT3 activation in macrophages by mechanism that does not involve Nrf2.
  • DI also affecting S727 phosphorylation in WT cells, which manifested as increased phosphorylation in unstimulated cells and decreased induction in phosphorylation after LSP stimulation compared to DI-untreated cells.
  • Ikb- ⁇ has been shown to be induced by stimulation of IL17 receptor in different cellular systems. It was hypothesized that DI could be potentially used to regulate Ikb- ⁇ induction in keratinocytes. To test this hypothesis, human immortalized HaCat kereatinocyte cell line was used, and DI effect on IL17A-mediated Ikb- ⁇ induction was tested. Ikb- ⁇ induction increased during 4 h of IL17A stimulation and 12 h of DI pretreatment at non-toxic concentrations almost completely inhibited this induction (see e.g., FIG.4A). All tested DI concentrations were also effective to inhibit STAT3 activation.
  • the cell viability was determined by flow cytometry (see e.g., FIG.4B).
  • flow cytometry see e.g., FIG.4B.
  • human primary keratinocytes were generated. In primary cells only long form of Ikb- ⁇ has been induced by IL17. Presence of short form of Ikb- ⁇ might reflect a donor specific condition. Induction of the long form of Ikb- ⁇ was inhibited by DI treatment (see e.g., FIG.4C). Viability of primary keratinocytes was not affected by DI treatment (see e.g., FIG.4D). In line with Ikb- ⁇ down-regulation, expression of IL-17A signature genes including Lcn2 and S100a9 was also inhibited as detected by qPCR (see e.g., FIG.4E).
  • NFKBIZ was identified as a new psoriasis susceptibility locus and Ikb- ⁇ has been shown to act as a direct transcriptional activator of TNF ⁇ /IL17-inducible psoriasis-associated genes and as a key driver in development of psoriasis.
  • DI To characterize effect of DI in the pathogenesis of psoriasis a mouse model of psoriasis- like skin inflammation (induced by topical application of the Toll-like receptor (TLR) agonist imiquimod) and which in many ways models human psoriasis was used. DI was administered to mice daily, which led to significant improvement of the psoriatic pathology (see e.g., FIG.5A).
  • the same daily regimen of DI administration was used in vivo in the EAE model of the mice and found that disease was ameliorated (see e.g., FIG.5B).
  • DI selectively inhibits growth of ABC subtype of DLBCL lymphoma
  • DMF Dimethyl fumarate
  • DMF DMF-induced STAT3 activation.
  • concentrations of DMF were effective to inhibit STAT3 activation. These data strongly support the notion that DMF might exert some of its physiological effects through inhibition of Ikb- ⁇ and STAT3 inhibition. Based on the obtained data it DMF can also be effective in treatment of ABC DLBCL lymphoma subtypes (see e.g., FIG.7).
  • DI Dimethyl itaconate
  • BMDMs selectively inhibits LPS-induced production of inflammatory cytokines such as IL-1 ⁇ , IL-6, IL-12, but not TNF [1].
  • cytokines such as IL-1 ⁇ , IL-6, IL-12, but not TNF [1].
  • TNF TNF
  • Transcriptional profile of DI treated cells shows signature typical for oxidative and xenobiotic/electrophilic stress.
  • DI-treated cells exhibited increased intracellular ROS production and drop in cellular glutathione levels, events typical for Nrf2-inducing agents.
  • a number of Nrf2-triggering agents modulate inflammatory conditions by down-regulating the NF- ⁇ B pathway [2], however, at concentrations effective to inhibit IL-6, DI did not show inhibition of NF- ⁇ B signaling. Therefore selective action of DI towards NF- ⁇ B primary response gene, Ikb ⁇ , which selectively regulates IL- 6, IL-12 but not TNF transcription, was tested.
  • Ikb ⁇ NF- ⁇ B primary response gene
  • DI TNF/IL-17-inducible psoriasis-associated genes such as Defb4, S100a7a, S100a9a, or Lcn2 and as a key driver in development of psoriasis [4].
  • DI the effect of DI on Ikb ⁇ -induction in keratinocytes was tested. It was shown that DI effectively inhibits Ikb ⁇ in IL-17A stimulated keratinocytes and down-regulates expression of Ikb ⁇ -target genes.
  • DI is capable to ameliorate psoriatic pathology associated with murine model of psoriasis and down-regulates major Ikb ⁇ target genes in skin tissue. Based on these data it is suggested that DI can be a therapeutic option for treatment of Ikbz/IL17 associated diseases.
  • FIG.8 shows (A) Structure of DI. (B) Comparison of transcriptional profiles of cKeap KO BMDMs and BMDMs treated with 250 ⁇ M DI for 12 h. (C)
  • Nrf2 expression and expression of Nrf2-targets (Nqo1, HO-1) in BMDMs treated as in B.
  • D Analysis of intracellular DI uptake and identification of DI-glutathione adduct.
  • D ROS production measured by detection of CM-H2DCFDA in BV2 cells treated with DI as in (B).
  • F Glutathione depletion and GSH/GSSG ratio (G) in BMDMs treated with DI as in (B).
  • NAC N-acetylcysteine neutralizes effect of DI on cytokine production.
  • BMDMs were treated with 250 ⁇ M DI and stimulated with LPS for 12 h. In some samples NAC was added simultaneously with DI.
  • FIG.9 (A) BMDMs were treated with 250 ⁇ M DI for 12 h and stimulated with LPS. Loss of IRAK1 detection upon LPS refers to its K63 ubiquitination [2] that correlates with activation. DI affected phosphorylation of IKK in time-dependent manner. (B) Degradation of Ikb ⁇ is also not affected by DI treatment. (B) BMDMs were treated with DI as in A and fixed, permeabilized and stained for p65, F-Actin and nuclei. DI pretreatment does not inhibit nuclear translocation of p65 upon LPS stimulation.
  • FIG.10 (A) Detection of Ikb ⁇ expression in BMDMs treated with DI for 12 h and stimulated with LPS. (B) Dose dependent effect of DI on cytokine production in BMDMs pretreated with DI for 12h and stimulated with LPS for 4h. (C) Dose dependent inhibition of Ikb ⁇ expression in in cell treated as in B and stimulated with LPS for 1h. (D) Nfkbiz mRNA expression in Ikb ⁇ protein expression in cells treated as in (C).
  • BMDMs were treated with 10 mM DM for 12h and stimulated with LPS.
  • FIG.11 Ikb ⁇ expression in BMDMs treated with 250 ⁇ M DI for 12h and stimulated with LPS. In some samples N-acetylcysteine was added
  • FIG.12 Ikb ⁇ expression in primary mouse keratinocytes that were treated with DI for 12 h and then stimulated with IL-17A (100 ng/ml).
  • B Viability of mouse keratinocytes treated as in (A).
  • C qPCR analysis of gene expression in mouse keratinocytes treated as in (A).
  • D Ikb ⁇ expression in primary human keratinocytes that were treated with DI for 12h then stimulated with IL-17A (100 ng/ml).
  • E Viability of mouse keratinocytes treated as in (D).
  • F qPCR analysis of gene expression in mouse keratinocytes treated as in (D).
  • FIG.13A Bl6 mice were injected i.p. with a dose of DI and imiquimod (IMQ) was applied topically on ear skin daily for 7 days. Sections of imiquimod-treated ears from mice following 5 d of treatment are shown. FIG.13B qPCR analysis of gene expression in skin tissue of mice treated as in (A).
  • DI administration in vivo ameliorates IL-17/I ⁇ B ⁇ -driven skin pathology in the mouse model of psoriasis, highlighting therapeutic potential of this regulatory pathway.
  • the present results demonstrate that targeting the DI-I ⁇ B ⁇ regulatory axis can serve as an important new strategy for the treatment of I ⁇ B ⁇ -mediated autoimmune diseases.
  • BMDMs bone-marrow derived macrophages
  • Nrf2 protein levels as well as the protein levels of its classical target genes increased during 12 hour DI treatment (see e.g., FIG. 14B).
  • Endogenous itaconate also induced Keap1-Nrf2 response, as Nrf2 protein levels upon LPS activation were higher in WT macrophages than in Irg1-/- (see e.g., FIG. 14C).
  • transcriptional signatures of DI treatment matched those of the Keap1 knockout macrophages (see e.g., FIG.15A) indicating induction of global electrophilic stress response.
  • DI can readily act as an electrophile in Michael reaction (see e.g., FIG.15B) and trigger electrophilic stress, which is commonly controlled via glutathione (GSH) buffering.
  • GSH glutathione
  • covalent conjugation with GSH was described for fumarate, a metabolite typically accumulated in cells with mutated fumarate hydratase.
  • Analysis of the cell media from DI-treated macrophages suggested that it was uptaken from media and showed significant peak at retention time 14.4 min with the m/z of 464.1334 that was accumulating during incubation with DI and
  • DI-GSH diester of methylsuccinated GSH
  • FIG.14D, FIG. 14E, FIG.15C, and FIG.15D DI-GSH origin was confirmed using synthesized 13C5- labeled DI for cell treatment (see e.g., FIG.15E).
  • the reactivity with GSH was also observed for natural itaconate; methylsuccinated GSH (Ita-GSH, see e.g., FIG. 14D) was detected in LPS-stimulated macrophages that correlated with itaconate production and was absent in Irg1-/- cells (see e.g., FIG.14F).
  • TNFA is induced during primary transcriptional response to TLR stimulation, while Il6 is a product of the secondary transcriptional responses.
  • I ⁇ B ⁇ transcriptional response to TLR activation is I ⁇ B ⁇ , encoded by the Nfkbiz gene (see e.g., FIG.16A, FIG 17A, and FIG.17B).
  • DI completely abolished LPS-induced induction of I ⁇ B ⁇ protein and inhibited its target genes (Il12b, Edn1, etc.) in both BMDMs and human blood monocytes (see e.g., FIG.16B, FIG 17C, FIG 17D, FIG 17E, FIG 17F, and FIG.17G).
  • target genes Il12b, Edn1, etc.
  • DI did not inhibit I ⁇ B ⁇ degradation and upstream signaling in response to LPS nor did it prevent LPS-mediated p65 nuclear translocation (see e.g., FIG 17H, FIG 17I, and FIG.17J). Whether NAC/EtGSH-mediated reversal of the DI effect on IL-6 was associated with recovery of I ⁇ B ⁇ protein induction was then tested.
  • Nfkbiz mRNA levels were not changed by DI, affecting I ⁇ B ⁇ protein induction at the post-transcriptional level (see e.g., FIG.16D, FIG 16E, and FIG.18A).
  • BSO buthionine sulfoximine
  • Nrf2 is involved in the DI inhibition of I ⁇ B ⁇ synthesis was tested. Nrf2-deficient BMDMs did not alleviate DI- mediated I ⁇ B ⁇ and IL-6 inhibition (see e.g., FIG.20A, FIG.20B, and FIG.21A).
  • RNA-seq in Nrf2-/- and WT BMDMs were performed and the genes that were differentially expressed upon DI treatment were analyzed (see e.g., FIG.21E).
  • Most differentially DI-regulated pathways included upregulated integrated stress response pathways (Atf3, Atf4 and Eif2ak3/PERK etc.), while downregulating interferon (IFN) response pathway (Isg15 etc.) (see e.g., FIG.20C and FIG.21F).
  • Nrf2-independent transcriptional signature of DI treatment was compared to the publicly available dataset that profiled WT and Atf3-/- macrophages at their basal states. Strikingly, highly statistically significant overlap between genes regulated by ATF3 and genes regulated by DI were found (see e.g., FIG.20D), suggesting that DI action might be mediated by ATF3.
  • ATF3 protein was upregulated by DI treatment even on Nrf2-/- background (see e.g., FIG.20E). Furthermore, Atf3-/- cells restored I ⁇ B ⁇ protein levels upon DI treatment and significantly increased IL-6 production in DI-treated Atf3-/- cells compared to WT (see e.g., FIG.20F and FIG.20G). In Atf3-/- macrophages, DI failed to increase eIF2 ⁇ phosphorylation, even though it still induced Nrf2 response (see e.g., FIG.21G and FIG.21H).
  • DI-mediated ATF3 expression was efficiently decreased by co-treatment with NAC or EtGSH in both mouse macrophages and human monocytes (see e.g., FIG.21I, FIG.21J, and FIG.21K).
  • Endogenous itaconate also efficiently induced ATF3 response: when comparing WT and Irg1-/- BMDMs tolerized in the presence of BSO and then restimulated, ATF3 was induced in WT cells but not in the Irg1-/- (see e.g., FIG.20H).
  • I ⁇ B ⁇ also plays major role outside the macrophage context: it is induced upon IL-17 treatment of epithelial cells and orchestrates
  • I ⁇ B ⁇ induction of I ⁇ B ⁇ was inhibited by DI in primary mouse and human keratinocytes (see e.g., FIG.22A, FIG.22B, and FIG.23).
  • I ⁇ B ⁇ target genes such as Defb4, S100a7a, Lcn2 and S100a9 in mouse and human keratinocytes were analyzed. As expected, expression of these genes was downregulated by DI in correlation with I ⁇ B ⁇ protein levels (see e.g., FIG.22C and FIG. 22D).
  • C57BL/6N WT were from Charles River Laboratories. Irg1-/- mice were previously published. Nrf2-/- mice (cat# 017009) and control WT C57BL/6J mice (cat# 000664) were purchased from Jackson Laboratory. Nfkbiz-/- mice described were provided by Prof. Shizuo Akira, sex matched animals were used in experimnets. p62- deficient mice were provided by Prof. Herbert W. Virgin (Department of Pathology and Immunology, Washington University School of Medicine, USA). Mice were maintained at Washington University under specific pathogen-free conditions in accordance with Federal and University guidelines and protocols approved by the Animal Studies
  • Femurs and tibias from Hmox1lox/- and control LyzMcre/creHmox1lox/- described were provided by Dr. Miguel P. Soares.
  • Femurs and tibias from Atf3-/- mice as described and control C57BL/6 WT mice were provided by Dr. Tsonwin Hai. Mice used for the study were 6-12 weeks old. If not indicated otherwise, female mice were used.
  • BMDMs Bone marrow-derived macrophages
  • BMDM were prepared from 6- to 12-week-old mice as described1 and cultured in RMPI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin and mouse recombinant M- CSF (20 ⁇ g/mL, Peprotech). For experiments, cells were seeded at concentration 106 cells/mL in tissue culture plates of various formats.
  • FBS fetal bovine serum
  • M- CSF mouse recombinant M- CSF
  • the cells were treated with DI (250 ⁇ M (unless stated otherwise), cat# 592498, Sigma), DMF (50 ⁇ M (unless stated otherwise), cat# 242926, Sigma), 3-(ethoxycarbonyl)but-3-enoic acid (3MI, 5 ⁇ M, Aris Pharmaceuticals Inc.), 4-ethoxy-2-methylene-4-oxobutanoic acid (MI, Aris
  • LPS lipopolysaccharide
  • IFN- ⁇ 50 ng/mL
  • Peprotech Peprotech
  • cells were treated with ⁇ -tocopherol (AT, 10 ⁇ M; Sigma),
  • MitoTEMPO (MT, 500 ⁇ M; Sigma), N-acetylcysteine (NAC, 1 mM, Sigma), ethylester of GSH (EtGSH; 1 mM, Santa Cruz) or buthionene sulfoximine (BSO, 500 ⁇ M, Sigma) alone or in combination with DI for 12 h.
  • BMDMs were treated with DI for 12 h and bafilomycin A (BafA, 100 nM, Sigma) or MG132 (10 ⁇ M, Selleckchem) were added 30 min before subsequent LPS stimulation.
  • BV2 microglial cell line was a kind gift from Prof. Herbert W. Virgin (Department of Pathology and Immunology, Washington University School of Medicine, USA). BV2 cells were maintained in DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate and 100 U/mL penicillin-streptomycin.
  • RNA was extracted with oligo-dT beads (Invitrogen), and libraries were prepared and quantified as described. All RNA-seq experiments generated in the study were performed in n 2 independent cultures.
  • Pre-ranked gene set enrichment analysis (GSEA) was done using fgsea R package.
  • GSEA Gene Expression Omnibus
  • WT and Nrf2-/- BMDMs genes were ranked according signal to noise statistic, only top 10,000 genes ordered by mean expression were considered. MSigDB C2 and H gene set collections were used.
  • Keap1 conditional KO (KpCKO)(GSE71263) and Atf3-/- (GSE61055) datasets differential expression analysis was carried out using limma package, genes were ranked by the
  • test statistics and P were calculated using pre-ranked gene set enrichment analysis method fgsea package with 200,000 gene-set permutations.
  • Non-specific binding was blocked with 5% skim milk (or 5% BSA when phospho-proteins were analyzed), and membranes were probed with primary antibodies specific to Nrf2 (#12721), HO-1 (#70081), I ⁇ B ⁇ (mouse specific, #93726), I ⁇ B ⁇ (#9244), ATF3 (#D2Y5W), IRAK1 (#4504; sensitivity of IRAK1 detection diminishes upon IRAK1 K63 ubiquitination), phospho-IKK (Ser176/180, #2697), p62/SQSTM1 (#5114), phospho-eIF2 ⁇ (Ser51, #9721), eIF2 ⁇ (#5324) from Cell Signaling; GAPDH (sc-25778), I ⁇ B ⁇ (sc-1643), ATF3 (sc-188), SDHA (sc-166909) from Santa Cruz; NQO1 (ab28947) from Abcam, followed by incubation with anti-rabbit-HRP (1
  • Membranes were exposed to X-Ray films (Research Products International Corp.) and developed using SRX-101A film processor (Konica Minolta). GAPDH run on the same blot was used as loading control. After scanning of original films image brightness of some blots was adjusted and bands were cropped using ImageJ. Densitometry was performed using ImageJ. DI detection with gas-chromatography-mass spectrometry (GCMS)
  • GCMS analysis was performed using a Thermo Trace 1300 GC equipped with a 30m DB-35MS capillary column connected to a Thermo TSQ Quantum MS operating under electron impact (El) ionization at 70 eV.1 ⁇ L of sample was injected in splitless mode at 270 °C, using helium as the carrier gas at a flow rate of 1 mL min-1.
  • the GC oven temperature was held at 100 °C for 3 min and increased to 240 °C at 3.5° min-1.
  • the MS source and quadrupole were held at 230 °C and 280 °C, respectively, and the detector recorded ion abundance in the range of 30– 800 m/z.
  • Bone marrow-derived macrophages were seeded in 96-well plates at 105 cell per well for all analyses. After treatment, media was removed from the wells and the cells were washed 3 ⁇ with PBS (37 °C) and immediately placed on dry ice. The frozen cells or media were stored at -80 °C until extraction. Cell extracts were prepared by adding 180 ⁇ L of 70/30 Ethanol/H2O solution at 70 °C with 300 ng/mL 13C515N1 d5-Glutamate as the internal standard.
  • HRAM data was acquired using a QExactiveTM Orbitrap mass spectrometer (Thermo Fisher Scientific), which was equipped with a heated electrospray ionization source (HESI-II), operated in negative electrospray mode. Ionization source working
  • the heater temperature was set to 300°C
  • ion spray voltage was set to 3500 V.
  • An m/z scan range from 70 to 700 was chosen and the resolution was set at 70,000.
  • the automatic gain control target was set at 1e6 and the maximum injection time was 250 ms. Instrument control and acquisition was carried out by
  • Solvents A and B are water w/0.1% formic acid and acetonitrile, respectively.10 minute method time with a gradient from 10% B to 40% B over 5 minutes. Samples were loaded at 10% B.
  • E. coli ita23 (provided by Klamt Lab, Max Planck Institute) was allowed to grow in 12C-glucose-LB broth (10 mL of 10 g/L bacto-trypsin, 5 g/L yeast extract, 10 g/L NaCl, 0.28 g/L CaCl2, 125 mg/L kanamycin and 0.2% (w/v) of 12C- glucose) overnight at 30 oC and 210 rpm (until OD420: 2.6).
  • the production of itaconic acid was next initiated by dilution of 100 ⁇ L of the above culture into 250 mL of a 13C- glucose minimal media (5.0 g/L K2HPO4, 3.5 g/L KH2PO4, 3.5 g/L (NH3)NaHPO4, 0.25 g/L MgSO4, 11.3 mg/L CaCl2, 1.5 g/L glutamic acid, 0.5 mg/L thiamine, 25 mg/L kanamycin, 1 mL trace element solution and 0.4% 13C-glucose).
  • a 13C- glucose minimal media 5.0 g/L K2HPO4, 3.5 g/L KH2PO4, 3.5 g/L (NH3)NaHPO4, 0.25 g/L MgSO4, 11.3 mg/L CaCl2, 1.5 g/L glutamic acid, 0.5 mg/L thiamine, 25 mg/L kanamycin, 1 mL trace element solution and 0.4% 13C-glucose).
  • the trace element solution consists of 1.6 g/L FeCl3, 0.2 g/L CoCl26H2O, 0.1 g/L CaCl2, 0.2 g/L ZnCl2 4H2O, 0.2 g/L NaMoO4, 0.05 g/L H3BO3.
  • the bacteria were allowed to grow for 6 days until the OD420 reached 1.9-2.1.
  • the cells were pelleted by centrifugation for 30 min at 14,000g and 4 oC. The supernatant was collected and lyophilized to afford a white powder (3.78 g).
  • BMDMs were plated 2 ⁇ 106 cells per well in 6-well plates.
  • Cells were either (1) treated with DI (250 ⁇ M) for 12 h and then stimulated with LPS (100 ng/mL) for 1 h, or (2) only stimulated with LPS (100 ng/mL) for 1h, or (3) only treated with DI (250 ⁇ M) for 12 h, or (4) neither treated with DI nor LPS.
  • Cells were then washed 3 ⁇ with PBS and lysed in 200 ⁇ L of urea buffer (8 M urea, 75 mM NaCl, 50 mM Tris pH 8.0, 1 mM EDTA). Lysates were then cleared by centrifugation at 20,000g and protein concentrations were determined by BCA assay (Pierce).15 ⁇ g of total protein per sample were processed further. Disulfide bonds were reduced with 5 mM
  • dithiothreitol and cysteines were subsequently alkylated with 10 mM iodoacetamide.
  • Samples were diluted 1:4 with 50 mM Tris/HCl (pH 8.0) and sequencing grade modified trypsin (Promega) was added in an enzyme-to-substrate ratio of 1:50. After 16 h of digestion, samples were acidified with 1% formic acid (final concentration). Tryptic peptides were desalted on C18 StageTips according to and evaporated to dryness in a vacuum concentrator. Desalted peptides were labeled with the TMT10plex mass tag labeling reagent according to the manufacturer’s instructions (Thermo Scientific) with small modifications.
  • TMT10plex reagent 0.2 units of TMT10plex reagent was used per 15 ⁇ g of sample. Peptides were dissolved in 30 ⁇ l of 50 mM Hepes pH 8.5 solution and the TMT10plex reagent was added in 12.3 ⁇ l of MeCN. After 1 h incubation the reaction was stopped with 2.5 ⁇ l 5% hydroxylamine for 15 min at 25°C. Differentially labeled peptides were mixed for each replicate and subsequently desalted on C18 StageTips and evaporated to dryness in a vacuum concentrator.
  • the peptide mixtures were fractionated by Strong Cation Exchange (SCX) using StageTips as previously described with slight modifications. Briefly, one StageTip was prepared per sample by 3 SCX discs (3M, #2251) topped with 2 C18 discs (3M, #2215). The packed StageTips were first washed with 100 ⁇ l methanol and then with 100 ⁇ l 80% acetonitrile and 0.2% formic acid. Afterwards they were
  • the first fraction was eluted with 50 ⁇ l 50 mM NH4AcO; 20% MeCN (pH ⁇ 7.2), the second with 50 ⁇ l 50 mM NH4HCO3; 20% MeCN (pH ⁇ 8.5) and the sixth with 50 ⁇ l 0.1% NH4OH; 20% MeCN (pH ⁇ 9.5).200 ⁇ l of 0.2% acetic acid was added to each of the 3 fractions and they were subsequently desalted on C18 StageTips as previously described and evaporated to dryness in a vacuum concentrator. Peptides were reconstituted in 10 ⁇ l 0.2% formic acid. Both the
  • MaxQuant generated corrected TMT intensities were normalized such that at each condition/time point the corrected TMT intensity values added up to exactly 1,000,000, therefore each protein group value can be regarded as a normalized microshare (this was done separately for each TMT channel for all proteins that made the filter cutoff in all the TMT channels). After that a pseudocount of 1 was added to each intensity value in order to account for the noise level and make the fold change calls more robust for small intensity values. Finally, all values were log2 transformed and the average of the log2 values for all replicates per condition was established (if replicate samples were present).
  • BMDMs were grown in 12-well plate, 106 cells per well and treated with DI (250 ⁇ M) for 10 h. The cells were then washed 3 ⁇ with methionine deficient media and incubated 1 h without methionine in presence of 200 ⁇ M DI for 1h. After that L-azidohomoalanine (Click-iT® AHA, C10102, Invitrogen) was added directly to cell media to final concentration 50 ⁇ M. The cells were then stimulated with LPS for 1 h. In some samples, cells were treated with puromycin 5 ⁇ g/mL 2 h before LPS stimulation to block translation.
  • lysis buffer 50 mM Tris-HCl, pH 8, 1% SDS
  • protease inhibitor cocktail PMSF and Na3VO4 (Santa Cruz). Lysates were incubated on ice for 30 min, sonicated and cleared by centrifugation 13,000g for 5 min at 4 oC. Total protein concentration was determined using RC/DCTM Protein Assay (Biorad).30 ⁇ g of protein was used for downstream reaction with 40 nM biotin-alkyne (B10185, Invitrogen) and reaction was carried out in Click-iT® Protein Reaction Buffer Kit (C10276, Invitrogen) according to manufacturer's protocol.
  • Proteins were separated on 4%–20% polyacrylamide gradient gels (BioRad) and biotinylated proteins were detected by western blot with streptavidin-HRP conjugate (1:1000, #554066, BD Pharmingen). Membrane was striped using 0.2 M NaOH and reprobed to detect I ⁇ B ⁇ (see Western blot analysis section).
  • Total GSH concentration in cells was determined by GSH/GSSH Ratio Detection Assay Kit (Abcam) according to manufacturer protocol. Briefly, 106 BMDMs were lysed in 100 ⁇ L of 0.5% NP-40 in PBS, pH 6. Samples were deproteinized using trichloroacetic acid and neutralized by addition of 1M NaHCO3 to achieve pH 4-6. Collected extracts were diluted with supplied assay buffer and directly used for GSH measurement.
  • Cytokines in cell supernatants were analyzed using DuoSet® ELISA kits according to manufacturer protocol (R&D Systems). The supernatants from BMDMs were diluted 1:4, from human blood monocytes 1:3.
  • RNA isolation and quantitative real-time PCR [00259] RNA from cultured cells was isolated using a Total RNA I kit (OMEGA). RNA from mouse ear skin was extracted using RNAeasy mini kit (Qiagen) after tissue disruption with sterile zirconium beads on a MagNA Lyser (Roche). Isolated RNA was reverse transcribed using AffinityScript Multi-Temp reverse transcriptase (Agilent Technologies) according to the manufacturer’s protocol. Reactions were performed in 96-well plates using a SYBR Green PCR Master mix (Thermo Fisher Scientific) using a LightCycler® 96 or LightCycler® 480 (Roche Diagnostics).
  • Mouse Nfkbiz 3'UTR Lenti-reporter-GFP (#MT-m64048) vector or pLenti-UTR-GFP-Blank vector (#m014) were purchased from Applied Biological Materials.
  • Opti-MEM medium Invitrogen
  • 18 ⁇ g of psPAX2 gift from Didier Trono, Addgene plasmid #12260
  • 13 ⁇ g of pCMV-V-SVG gift from Bob Weinberg, Addgene plasmid #8454
  • 20 ⁇ g of lentiviral construct and 105 ⁇ L of polyethylenimine (1 mg/mL; 25 kDa; linear form; Polysciences).
  • HEK-293T cells ATCC
  • DMEM fetal bovine serum
  • penicillin-streptomycin 100 U/mL penicillin-streptomycin in 150-cm2 tissue culture flask.48 h later, virus- containing medium was filtered through 45 ⁇ m pore-size cellulose acetate filters and used directly for BV2 transduction in the presence of polybrene (8 ⁇ g/mL, Millipore).
  • media was changed to fresh and two days after the cells were selected with puromycin at 5 ⁇ g/mL.
  • BMDMs were seeded at eight-well multitest microscopy slides (MP Biomedicals). Cells were treated with DI (250 ⁇ M, 12 hours) and then stimulated with LPS (100 ng/mL, 30 min). The cells were fixed with 3% paraformaldehyde in PBS for 30 min and permeabilized with 0.1% Triton X-100 in PBS for 15 min. After washing with PBS, samples were blocked with 1% BSA for 15 min and subsequently labeled with p65-specific antibody (1:50, #8242, Cell Signaling), followed by AF568-conjugated anti- rabbit secondary antibody (1:500, #A11011, Thermo Fisher Scientific) in PBS containing 1% BSA.
  • the cells were washed with PBS and mounted in 50% (w/v) glycerol in PBS, pH 8.5 containing DAPI (1 ⁇ g/mL, Sigma) to label nuclei.
  • Images of random fields of view were acquired using a Leica DMi8 confocal microscope (Leica Microsystems) equipped with a HC PL APO 40x/1.3 oil immersion objective and exported with LAS AF Lite software (Leica).
  • PBMCs mononuclear cells
  • Imiquimod (IMQ, Imiquimod Cream 5%, Perrigo. Co.) was applied daily to mice on both ears, ( ⁇ 5 mg per ear) for 7 days.
  • DI was administered via the intraperitoneal route at 20 mg /500 ⁇ L sterile PBS per mouse one day prior to IMQ application and daily thereafter for 7 days.
  • mice were euthanized and ears were used for RNA extraction or histological analysis by performing H&E staining on 7 ⁇ m thick sections following paraffin embedding. Average ear thickness in each sample was quantified from images obtained at the identical settings using ImageJ. Thickness at 5 equidistant places in each image was quantified and mean of these values was used to represent results in each mouse.
  • DI was administered to mice via the intraperitoneal route at 20 mg /500 ⁇ L sterile PBS per mouse either once per day for total length of 4 days (DI daily) or every 2 h, 3 times in total (DI overdose).
  • DI daily last injection was 6 h before the tissue harvest
  • DI overdose protocol last injection was 2 h before harvest.
  • Mice were euthanized and heart ( ⁇ 50 mg) and liver tissue ( ⁇ 200 mg) were harvested, washed in PBS and processed for mitochondria isolation with Mitochondria Isolation Kit for Tissue (#89801, Thermo Scientific) according to manufacturer's instruction.
  • cytoplasmic and mitochondrial fractions were diluted with reducing sample buffer and analyzed by western blot for SDH and GAPDH presence (see western blot analysis section).
  • SDH activity in isolated mitochondria was analyzed using SDH Activity Colorimetric Kit (#MAK197, Sigma). Isolated mitochondria were directly resuspended in SDH Assay Buffer.
  • protein concentration in each sample was determined using RC/DCTM Protein Assay (Biorad) and activity was normalized to protein concentration.
  • RNA-seq data have been deposited to Gene Expression Omnibus with access number GSE102190 and GSE110749.
  • the original mass spectra may be downloaded from MassIVE (http://massive.ucsd.edu) using the identifier: MSV000082101.
  • Source Data for the graphical representations found in all Figures and Extended Data Figures are provided. All other data that support the findings of this study are available from the corresponding author upon reasonable request.
  • Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription. Nat. Commun.7, 11624 (2016).
  • Muromoto, R. et al. IL-17A plays a central role in the expression of psoriasis signature genes through the induction of I ⁇ B- ⁇ in keratinocytes. Int. Immunol.28, 443– 452 (2016).
  • microglial ⁇ 7-acetylcholine nicotinic receptor is a key element in promoting neuroprotection by inducing heme oxygenase-1 via nuclear factor erythroid-2-related factor 2. Antioxid. Redox Signal.19, 1135–1148 (2013).

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Abstract

Un des divers aspects de la présente invention concerne la fourniture d'une composition contenant de l'itaconate, du malonate ou un de leurs dérivés et les utilisations de ladite composition. La présente invention concerne des procédés de traitement d'une maladie, d'un trouble ou d'états associés à Ikb-ζ consistant à administrer à un sujet une quantité thérapeutiquement efficace d'itaconate, de malonate ou d'un de leurs dérivés. Dans certains modes de réalisation, la quantité thérapeutiquement efficace réduit ou empêche une croissance tumorale, une inflammation ou une réponse immunitaire. Un autre aspect de la présente invention concerne un procédé de suppression de l'induction de Ikb-ζ consistant à administrer de l'itaconate, du malonate ou un de leurs dérivés.
EP18845621.4A 2017-08-14 2018-08-14 Procédés et compositions de traitement de maladies associées au cancer, à une inflammation ou à une réponse immunitaire Withdrawn EP3668497A4 (fr)

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